Enzymatic cycling assays for homocysteine and cystathionine

ABSTRACT

The present invention provides an enzymatic cycling assay for assessing the amount of homocysteine and/or cystathionine in a solution such as blood, blood derivatives, or urine. The assay comprises the steps of contacting the solution containing homocysteine and/or cystathionine to form a reaction mixture, with CBS, or a derivative thereof, L-serine, and CBL, or a derivative thereof, for a time period sufficient to catalyze the cyclical conversion of homocysteine form to cystathionine and the reconversion of cystathionine to homocysteine with the production of pyruvate and ammonia; determining the amount of homocysteine and/or ammonia present in the reaction mixture; and determining the amount of homocysteine and/or cystathionine present in the solution based on the amount of pyruvate and/or ammonia formed. Expression vectors and isolation procedures for CBS, or derivatives thereof, and CBL, or derivatives thereof, are also provided as well as test kits for carrying out the assay. In preferred embodiments, the assays can be conducted in 15 minutes or less, with a minimum of enzyme usage.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/012,762, filed Nov. 6,2001, now U.S. Pat. No. 6,635,438, which is a continuation-in-partapplication of U.S. patent application Ser. No. 09/704,036, filed Nov.1, 2000 now U.S. Pat. No. 6,664,073 which claimed benefit fromProvisional Patent Applications Ser. No. 60/163,126, filed Nov. 2, 1999and Ser. No. 60/203,349 filed May 10, 2000, and the content andteachings of each of these previous applications is incorporated hereinby reference.

SEQUENCE LISTING

A compliant Sequence Listing containing 21 sequences in the form of acomputer readable ASCII file in connection with the present inventionwas filed in U.S. Ser. No. 10/012,762 and is incorporated herein byreference. Applicants request that this earlier-filed CRF be used as theCRF for this application. A paper copy of this sequence is includedherein and is identical to this previously filed CRF.

BACKGROUND OF THE INVENTION

High levels of homocysteine in human plasma are correlated withincreased risks for coronary heart disease, stroke, arteriosclerosis,and other diseases. As a result, it is desirable to screen the generalpopulation for elevated amounts of this amino acid. To make wide-scaletesting for homocysteine feasible, new and less expensive assays need tobe developed.

Plasma homocysteine is routinely measured by high-pressure liquidchroma-tography (HPLC) and gas chromatography/mass spectrometry (GC/MS)at a cost of over $100 per assay, making these physical separationmethods too costly for a population-wide study. For example, urine orblood samples can be prepared for amino acid chromatography, andL-homocysteine measured by HPLC separation and detection. Fiskerstrandet al. (Clin. Chem., 39:263-271 (1993)) describe a method of assayingL-homocysteine using fluorescent labeling of serum thiols, followed byHPLC separation and detection of the L-homocysteine derivative from thevarious other sulfur-containing compounds. However, such methods aretypically time consuming, costly, and not readily available to manylaboratories.

Indirect immunoassays for homocysteine have also been developed,however, these antibody methods are still relatively expensive at about$24 per test. One particular indirect immunoassay enzymatically convertshomocysteine to adenosyl homocysteine and the amount of adenosylhomocysteine is determined by a competitive ELISA (enzyme linkedimmunoassay), using an anti-adenosyl homocysteine antibody (for example,see U.S. Pat. No. 5,827,645, the content of which is incorporated hereinby reference).

Indirect enzyme assays have been developed for the quantitation ofL-homocysteine. For example, the enzyme S-adenosyl-homocysteinehydrolase and adenosine are added to a test sample. The resultingconcentration, or change in the concentration, of adenosine in thereaction mixture is used as an indicator for the initial concentrationof homocysteine in the sample.

Direct enzyme assays have also been reported for measuring homocysteine.Typically, these protocols irreversibly convert homocysteine to othercompounds that are quantifiable. For example, the enzyme homocysteinedehydratase has been used to remove the sulfhydryl group fromhomocysteine. The concentration of the removed sulfhydryl moiety is thenmeasured. A major drawback with this and other enzyme assays forhomocysteine is that the enzymes employed react with other sulfurcontaining amino acids and compounds related to homocysteine, leading toa high and inconsistent background and measurements of homocysteine fromplasma that are inaccurate.

Enzymatic (or enzymic) cycling assays have been reported for a verysmall number of analytes. In an enzymatic cycling assay two or moreenzymes activities are used which recycle substrate and do notirreversibly convert the measured compound. Instead the “compound” isused catalytically to control the rate of conversion to the quantitatedcompound in the assay. As a result, the analyte of interest remains in asteady-state concentration which is low enough to create a pseudo-firstorder rate of reaction. The steady-state concentration of the analyte isthereby linearly related to the rate of the overall assay. By measuringreaction rates, the amount of the analyte is easily determined.Enzymatic cycling assays are sometime called “amplification” assays,because the methods typically increase the sensitivity of measurementfor an analyte by 100- to 1000-fold. The amplification in measurement isa direct result of not reducing the steady-state concentration of thecompound. No enzymatic cycling assay has been reported for measuringhomocysteine.

The present invention provides an enzymatic cycling assay forhomocysteine and/or cystathionine which is less expensive, and providesa higher sample throughput than the diagnostic assays currentlyavailable. Further, the invention provides methods and vectors for therecombinant production of enzymes which can be used in the production ofassay reagents and test kits for assessing the amount of homocysteineand cystathionine in a sample.

SUMMARY OF THE INVENTION

The present invention provides an enzymatic cycling assay method forassessing the amount of homocysteine and/or cystathionine in a solution.The assay takes advantage of the reaction of homocysteine and L-serineto form cystathionine by the enzyme cystathionine β-synthase (CBS), or aderivative thereof, and the enzymatic conversion by cystathionineβ-lyase (CBL) of cystathionine to homocysteine, pyruvate and ammonia.The assay provides a steady-state concentration of the homocysteineand/or cystathionine which is linearly related to the rate of theoverall reaction. The amount of homocysteine and/or cystathioninedetermined in a sample is based on the amount of pyruvate and/or ammoniawhich is formed or the amount of serine removed from the reactionmixture. Solutions which can be tested using the assay of the presentinvention can include laboratory samples of blood, serum, plasma, urine,and other biological samples. Additionally, any other liquid sample canbe tested.

In one embodiment, the present invention provides a method for assessingthe amount of homocysteine and/or cystathionine in a solution comprisingthe step of:

(a) contacting the solution containing homocysteine and/or cystathionine(either before and/or after performing a disulfide reduction step) toform a reaction mixture, with CBS, or a derivative thereof, L-serine andCBL, or a derivative thereof, for a time period sufficient to catalyzethe cyclical conversion of homocysteine to cystathionine, and thereconversion of cystathionine to homocysteine with the production ofpyruvate and ammonia;

(b) determining the amount of pyruvate and/or ammonia present in thereaction mixture; and

(c) assessing the amount of homocysteine and/or cystathionine present inthe solution based on the amount of pyruvate and/or ammonia formed.

More particularly, the method provides for the assessment of the amountof homocysteine by the addition of the inexpensive amino acid L-serine.The amount of pyruvate present in the reaction mixture can be measuredin a number of ways. In one particular embodiment of the presentinvention lactate dehydrogenase (LDH), or a derivative thereof, and NADH(reduced nicotinamide cofactor), or a derivative thereof, are present inthe reaction mixture. LDH in the presence of NADH converts pyruvate tolactate with the oxidation of NADH to NAD+ (oxidized nicotinamidecofactor).

The oxidation of NADH to NAD+ can be measured by a number of methodsknown in the art including monitoring the reaction mixture at 340 nm.Production of NAD+ can also be monitored by the oxidation of a dye toproduce an observable color change. Dyes preferred for use in thepresent invention include, but are not limited to,5,5′-dithiobis(2-nitrobenzoic acid), 2,6-dichlorophenylindophenol,tetrazolium compounds, phenazine methosulfate, methyl viologen, orderivatives of any of these dyes. The amount of homocysteine and/orcystathionine present in the solution is based on the intensity of theobserved color compared to a standard curve constructed for samples ofknown concentration of the analyte.

In an alternative embodiment pyruvate oxidase, with horseradishperoxidase, in the presence of hydrogen peroxide and a chromogen areused to detect the amount of pyruvate present in the sample. SodiumN-ethyl-N-2-hydroxy 3-sulfopropyl) m-toluidine (TOOS) and otherN-ethyl-N-(2-hydroxy-3-sulfopropyl)-aniline derivatives are preferredchromogens for this colorimetric reaction. As above, the amount ofhomocysteine and/or cystathionine present in the sample is based on theintensity of the observed color compared to a standard curve constructedfor samples of known concentrations of the analyte.

The amount of homocysteine and/or cystathionine present in a solutioncan also be measured based on the amount of ammonia present in thereaction mixture. Methods for determining the concentration of ammoniain a solution are legion. In one particular embodiment of the presentinvention, the amount of ammonia is measured using a commonly availablestandard ammonia sensor.

In another embodiment, the present invention is directed to a method forassessing the amount of homocysteine and/or cystathionine in a samplecomprising the steps of contacting the solution with a reducing agentfor a time period sufficient to reduce substantially all homocysteineand other disulfides that are present in the solution to homocysteine.Treatment with a reducing agent can also act to release homocysteinewhich is attached to a protein and/or other molecules present in asolution through a disulfide bond. After reduction the solution is thencontacted with CBS, or a derivative thereof, L-serine, and CBL, or aderivative thereof, for a time period sufficient to catalyze thecyclical conversion of homocysteine to cystathionine and the conversionof cystathionine to homocysteine with the production of pyruvate andammonia. To assess the amount of homocysteine and/or cystathioninepresent in the solution the amount of pyruvate and/or ammonia present inthe reaction mixture can be determined as set forth above. Preferredreducing agents for use in the present invention include borohydridesalts and thiol reducing agents. Typical thiol reducing agentsappropriate for use in the present embodiment include dithioerythritol(DTE), dithiothreitol (DTT), β-mercaptoethanol (βME),tris-(carboxyethyl)phosphine hydrochloride (TCEP), or thioacetic acid,or any derivatives thereof, and the like.

In yet another embodiment for assessing the amount of homocysteineand/or cystathionine present in a solution, the solution can bepretreated with cystathionine γ-lyase (CGL), or a derivative thereof,for a time period sufficient to remove any cystathionine from thereaction mixture by the conversion of cystathionine to α-ketoglutarate.Following cystathionine removal the cystathionine γ-lyase is removedfrom the reaction mixture or destroyed. In a typical embodiment, thecystathionine γ-lyase is destroyed by heating the solution for a timeperiod sufficient to remove substantially its enzymatic activity. Thecystathionine γ-lyase can also be immobilized on an insoluble substrateor surface, such as, for example, a micro particle or bead, which can beeasily removed.

In still another embodiment of the present invention a method isprovided for assessing the amount of homocysteine and/or cystathioninepresent in a solution comprising the reaction of the solution withL-serine and CBS, or a derivative thereof, and CBL, or a derivativethereof, which have been immobilized on a solid surface. The solidsurface can be, for example, paper, filter paper, nylon, glass, ceramic,silica, alumina, diatomaceous earth, cellulose, polymethacrylate,polypropylene, polystyrene, polyethylene, polyvinylchloride, andderivatives thereof. The solid surface can be the sides and bottom ofthe test container or can be a material added to the test container. Ina preferred embodiment the solid surface comprises a bead which is addedto the test container.

The CBS, or derivative thereof, CBL, or derivative thereof, andcystathionine γ-lyase, or derivative thereof, useful in the presentinvention can be obtained as a crude extract from a cell. In oneembodiment of the present invention the cystathionine β-synthase (CBS),or derivative thereof, cystathionine β-lyase CBL, and/or cystathionineγ-lyase (CGL) are purified from human, yeast or bacterial cells. In aparticularly preferred embodiment of the present invention the geneswhich encode the enzymes are isolated or synthesized and are expressedas a recombinant protein in a host cell. It is particularly preferredthat a DNA sequence which encodes an affinity tag be added to the geneconstruct to aid in the purification and/or detection of therecombinantly produced enzymes. Recombinant methods can also be used toprovide fusion proteins which comprise the enzyme activities of CBS andCBL in a single protein. An affinity tag can also be included as part ofthe fusion protein construct to aid in the purification of the fusionprotein.

The present invention also provides as a method for assessing the amountof homocysteine in a sample an assay format which correlates the amountof homocysteine/transcription factor complex which is bound to aconsensus polynucleotide binding sequence. In a particular embodimentthe method comprises contacting the sample with a reducing agent for atime period sufficient to release homocysteine from any associatedprotein; contacting the reduced homocysteine with a homocysteinemetabolite binding transcription factor under conditions conducive forcomplex formation, admixing the sample with a consensus polynucleotidesequence specifically recognized by the homocysteine/transcriptionfactor complex; and assessing from the amount ofhomocysteine/transcription factor complex bound to the consensuspolynucleotide sequence the amount of homocysteine present in thesample. Reducing agents which are applicable for use in the methodcomprise borohydride salt or thiol reducing agents includingdithioerythritol (DTE), dithiothreitol (DTT), β-mercaptoethanol,tris-(carboxyethyl)phosphine (TCEP), or thioacetic acid, or any salt ofeach. Homocysteine metabolite binding transcription factors include MetRof E. coli which recognizes a consensus polynucleotide sequence, forexample, the polynucleotide sequence as depicted in SEQ ID NO: 11(Marconi et al., Biochem. Biophys. Res. Commun., 175:1057-1063 (1991))or a derivative thereof.

Yet another embodiment of the present invention provides a test kitcomprising a container for holding the solution to be assessed for theamount of homocysteine and/or cystathionine, L-serine, CBS, or aderivative thereof, CBL, or a derivative thereof, and any buffers,auxiliary substances and solvents required to provide conditionsconducive to high enzyme activity. The test kit can further compriselactate dehydrogenase, or a derivative thereof, and NADH, or aderivative thereof. NADH can be measured directly at 340 nm or, a dyecapable of providing a color change when oxidized can be included. Thequantity of homocysteine and/or cystathionine is correlated with thechange in absorbance measure over time.

In a preferred embodiment the enzymes are provided immobilized to asolid support. The solid support can comprise the surface of thecontainer provided to hold the test sample or can be a bead or otherarticle added to the container. In an additional embodiment of thepresent invention, cystathionine γ-lyase can be provided as part of thetest kit to remove any cystathionine from the test solution prior to theenzymatic cycling assay. Substantially all of the activity of thecystathionine γ-lyase, or derivative thereof, must be removed from ordestroyed in the reaction mixture prior to the addition of the remainingcomponents for the enzymatic cycling of homocysteine.

It has been found that the preferred assay of the invention can becarried out in a relatively short period of time and with relativelysmall amounts of enzyme, giving an assay which has substantialcommercial advantages. For example, the preferred assay involvescreation of a reaction mixture including a homocysteine-containingsample, serine, CBS, CBL, lactate dehydrogenase, NADH and a reductantsuch as DTE or DTT, with the CBS/CBL ratio in the mixture being fromabout 1:1 to 25:1, more preferably from about 1:10, and most preferablyfrom about 2:1 to 5:1. Advantageously, it has been found that thereductant can be present along with the detection system (i.e., thelactate dehydrogenase and NADH) without deleteriously affecting theassay. Accordingly, the assay of the invention can be carriedout-without a separate reduction step so that total assay times arereduced. Thus, where a plurality of samples are to be assayed bysequentially creating a reaction mixture in a container (e.g., aspectrophotometric cuvette) made up of a sample, serine, CBS, CBL,lactate dehydrogenase, NADH and the reductant, and assessing the amountof pyruvate present in the reaction mixture by monitoring the productionof NAD+ over time, the time interval between the respectivereaction-creation steps may be as short as 10-50 seconds with the totalreaction time being up to about 20 minutes for a given sample. Morepreferably, the total reaction time for a given sample is up to about 15minutes, and still more preferably up to about 13 minutes. The totalnumber of samples assayed per hour may be about 15-30 for slowerinstruments, but as high as 200-400 for faster instruments. In thispreferred assay, a first reaction mixture comprising the sample, serine,lactate dehydrogenase, NADH and the reductant is prepared, with asuitable incubation period to permit liberation of a preponderance (andpreferably essentially all) of the homocysteine (total homocysteine)present in the bound, oxidized and/or free states in the sample.Thereafter, CBS and CBL are added to complete the reaction mixture andinitiate enzymatic cycling of homocysteine and/or cystathionine.

In another preferred embodiment, cystathionine can be used to make acalibrator(s) for the enzyme assay (since the enzymatic cycling assayinterconverts homocysteine and cystathionine). In other words, varyingknown levels of cystathionine can be used in the assay system to“calibrate” or “standardize” the assay and/or instrument which allowsfor quantitation of sample results. In this embodiment, a knownconcentration of cystathionine is added to a biological sample and thensubjected to the assay used for one of the other embodiments. Theresults will be used to establish a calibration line which will then beused to set the homocysteine line, due to the high degree of correlationbetween the two lines. Alternatively, known levels of cystathionine canbe used as a quality control measure, to insure that the assay isworking properly. In this quality control embodiment, these “known”levels of cystathionine are assayed as if they were unknown samples, andthe results are compared to their known (expected) values, in order toinsure that the assay system is functioning properly.

The preferred assays are carried out using isolated (purified) CBL andCBS enzymes having at least about 80% (and preferably at least about90%) sequence identity with the enzymes selected from the groupconsisting of SEQ ID Nos. 19 and 20. As used herein, “sequence identity”as it is known in the art refers to a relationship between two or moreprotein or polypeptide sequences or two or more polynucleotidesequences, namely a reference sequence and a given sequence to becompared with the reference sequence. Sequence identity is determined bycomparing the given sequence to the reference sequence after thesequences have been optimally aligned to produce the highest degree ofsequence similarity, as determined by the match between strings of suchsequences. Upon such alignment, sequence identity is ascertained on aposition-by-position basis, e.g., the sequences are “identical” at aparticular position if at that position, the nucleotides or amino acidresidues are identical. The total number of such position identities isthen divided by the total number of nucleotides or residues in thereference sequence to give % sequence identity. Sequence identity can bereadily calculated by known methods, including but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. N., ed., OxfordUniversity Press, New York (1988), Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York (1993); ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinge, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988), the teachings of which are incorporated herein by reference.Preferred methods to determine the sequence identity are designed togive the largest match between the sequences tested. Methods todetermine sequence identity are codified in publicly available computerprograms which determine sequence identity between given sequences.Examples of such programs include, but are not limited to, the GCGprogram package (Devereux, J., et al., Nucleic Acids Research, 12(1):387(1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.Biol., 215:403-410 (1990). The BLASTX program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 95% “sequence identity” to a reference nucleotide sequence, itis intended that the nucleotide sequence of the given polynucleotide isidentical to the reference sequence except that the given polynucleotidesequence may include up to 5 point mutations per each 100 nucleotides ofthe reference nucleotide sequence. In other words, in a polynucleotidehaving a nucleotide sequence having at least 95% identity relative tothe reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving a given amino acid sequence having at least, for example, 95%sequence identity to a reference amino acid sequence, it is intendedthat the given amino acid sequence of the polypeptide is identical tothe reference sequence except that the given polypeptide sequence mayinclude up to 5 amino acid alterations per each 100 amino acids of thereference amino acid sequence. In other words, to obtain a givenpolypeptide sequence having at least 95% sequence identity with areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total number of aminoacid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or the carboxy terminal positions of the referenceamino acid sequence or anywhere between those terminal positions,interspersed either individually among residues in the referencesequence or in the one or more contiguous groups within the referencesequence. Preferably, residue positions which are not identical differby conservative amino acid substitutions. However, conservativesubstitutions are not included as a match when determining sequenceidentity.

Reagents useful in the present invention include CBS, CBL, L-serine,LDH, NADH, DTE, βME, DTT, TCEP, thioacetic acid, and CGL. Concentrationsof CBS enzyme useful in the present invention range from about 0.1 toabout 100 KU/l. More preferably, the CBS concentration is from about 0.5to about 75 KU/l, and still more preferably from about 1 to about 50KU/l. Most preferably, the concentration of CBS is from about 1 to about30 KU/l. Concentrations of CBL enzyme useful in the present inventionrange from about 0.01 to about 100 KU/l. More preferably, CBSconcentration is from about 0.05 to about 50 KU/l, and still morepreferably, from about 0.1 to about 30 KU/l. Most preferably, the CBSconcentration is from about 0.1 to about 15 KU/l. L-serine may bepresent at a final concentration of from about 1 μM to about 50 mM. Morepreferably, the L-serine is added at a final concentration of from about10 μM to about 40 mM, and still more preferably at a final concentrationof from about 100 μM to about 20 mM. Most preferably, the finalconcentration of L-serine added is from about 0.2 mM to about 10 mM.When used in any embodiment, LDH is present in the reaction mixture at afinal concentration of from about 30 to about 5000 U/L. More preferably,the final concentration of the LDH present in the reaction mixture isfrom about 30 to about 3000 U/L, and still more preferably from about 50to about 2500 U/L. Most preferably, LDH is present in the reactionmixture at a final concentration of from about 100 to about 2000 U/L.Additionally, when NADH is used in any embodiment, the amount of NADHpresent in the reaction mixture can vary between about 0.1 mM to about 2mM. More preferably, NADH is present in the reaction mixture at a finalconcentration of from about 0.1 mM to about 1.5 mM, and still morepreferably between about 0.1 to about 1 mM. Most preferably, NADH ispresent in the reaction mixture at a final concentration of from about0.2 to about 0.8 mM. When DTE is used in the present invention, it canrange in final concentration from about 0.01 mM to about 100 mM. Morepreferably, concentrations of DTE will range from about 0.01 mM to about50 mM, and still more preferably from about 0.1 mM to about 25 mM. Mostpreferably, final concentrations of DTE will range from about 0.1 mM toabout 10 mM. Similarly, when DTT is used in the present invention, itcan range in final concentration from about 0.01 mM to about 100 mM.More preferably, concentrations of DTT will range from about 0.01 mM toabout 50 mM, and still more preferably from about 0.1 mM to about 25 mM.Most preferably, final concentrations of DTT will range from about 0.1mM to about 10 mM. Finally, when CGL is used in the present invention,it can range in final concentration from about 0.1 KU/l to about 100KU/l. More preferably, CGL will range from about 0.5 KU/l to about 75KU/l, and still more preferably from about 1 KU/l to about 50 KU/l. Mostpreferably, final concentrations of CGL will range from about 1 KU/l toabout 30 KU/l.

In some preferred embodiments, the buffer of Reagent 1 is TRIS, 250 mMat pH 8.4. However, when TRIS is used as the buffer the pH may rangefrom 7.0-9.0. More preferably, the pH ranges from 7.5-8.5 and still morepreferably from about 8.1-8.5. The use of increased concentration ofTRIS improves assay consistency by helping to maintain a more constantpH as it is a more concentrated buffer. Additionally, CBS and CBL aresubstantially more active in this preferred pH range, and especiallyfrom about pH 8.1-8.5.

The present invention also provides an assay which works equally wellwith turbid samples. Problems with turbidity are reduced by addinglipase and-cyclodextrin to R1. These help to decrease turbidity by thehydrolysis of triglycerides to glycerol and free fatty acids by lipaseand by the formation of complexes with the free fatty acids by theα-cyclodextrin. The addition of these two components helped to clear thereaction mixture before the addition of Reagent 3. Replacing lipase andα-cyclodextrin with EDTA provides another means of accurately analyzingturbid samples.

Another variation of the present invention tested the effects of varyingvolumes of the reagents. Such variations did not have a substantialeffect on the accuracy of the assay.

The present invention also tested the effects of different detergentsand concentrations thereof. For example, Genapol X-80 may be present inReagent 1 at a concentration between about 0.05-0.5%. More preferably,the concentration is between 0.1-0.5% and still more preferably betweenabout 0.2-0.4%. Of these, three concentrations (0.1%, 0.3%, and 0.5%)were tested with the 0.3% concentration providing the most accurateresults. Brij-35 was also varied in concentration in Reagent 1. Brij-35may be used with the present invention in R1 at a concentration betweenabout 0.01-0.5%. More preferably, this concentration ranges from about0.015-0.1%, and still more preferably from about 0.020-0.030%. Of theconcentrations tested (0.025%, 0.05%, 0.1% and 0.5%), the most accurateand consistent results were obtained using a concentration of 0.025%.

The present invention also tested replacing the reducing agent DTE withTris (2-carboxyethylphosphine) hydrochloride (TCEP) which is stable inpurified water for an extended period of time. Such a substitutionreduced the reagent blank reaction from about 12-15 mA/min to about 4-5mA/min which enables a greater degree of precision for this assay. WhenTCEP is used in the present invention, it may range in concentrationfrom about 6-53 mM. More preferably, this concentration ranges fromabout 10-45 mM and still more preferably from about 20-30 mM. In theconcentrations tested in this application, 26.44 mM provided the mostaccurate assay results.

The mixture comprising Reagent 3 was also varied in composition. Forexample, the Tris buffer was replaced with a phosphate buffer andglycerol was added to the buffer. The phosphate was effective atconcentrations ranging from about 50 mM to about 500 mM at pH 7.6. Theglycerol concentrations ranged from about 0.5% to 15.0% (v/v). Phosphateand glycerol were used to provide longer shelf-life for the enzymes,which was not possible with the TRIS buffer.

One of the advantages of the present invention is that it may be usedwith any of a number of instruments. For example, the assay was adaptedfor testing on two different instruments, the Hitachi 911 and theBeckman CX5. Notably, accurate and consistent results were obtained witheither machine. Additionally, the assay can be run using just 2 reagentsby mixing the components of R1 and R2 together in the correct ratiobased on the instrument requirements.

Finally, the present invention can be adapted for use with a singlecalibration point. Importantly, the results from single pointcalibration was just as accurate as a multi-point calibration curve.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts absorbance versus time plots obtained using aqueoussolutions of pyruvate (5-100 μM) demonstrating the quantitation ofpyruvate. Reagent components were those described in Table 1. andinstrument settings those described in Table 2;

FIG. 2 depicts a standard curve of absorbance values plotted versusknown quantities of pyruvate (0-100 μM). Assay time was 5 min.;

FIG. 3 depicts absorbance versus time plots obtained using aqueoussolutions of cystathionine (0-100 μM). Cystathionine was measured by theenzymic conversion of cystathionine to homocysteine, pyruvate andammonia by CBL;

FIG. 4 depicts cystathionine assay standard curves obtained usingaqueous solutions of cystathionine (0 to 100 μM). Reaction time waseither 5 or 20 min.;

FIG. 5 depicts absorbance versus time plots demonstrating theCBS/CBL/pyruvate oxidase/peroxidase cycling and signaling system.Cystathionine was dissolved in water to prepare standards atconcentrations between 1 and 100 μM;

FIG. 6 depicts absorbance versus time plots demonstrating theCBS/CBL/pyruvate oxidase/peroxidase cycling and signaling system.Homocysteine was dissolved in water to prepare standards atconcentrations between 0 and 100 μM;

FIG. 7 depicts a typical calibration curve obtained using aqueoussolutions of homocysteine (0-100 μM) using the CBS/CBL/PO/HRP cyclingand detection system;

FIG. 8 depicts typical absorption versus time plots obtained usinghomocysteine (0-100 μM) in aqueous solution using the CBS/CBL/LDH systemwithout dithiotreitol;

FIG. 9 depicts typical absorption versus time plots obtained usinghomocysteine (0-100 μM) in aqueous solution using the CBS/CBL/LDHcycling system with dithiotreitol;

FIG. 10 depicts a calibration plot of homocysteine concentration (0 to100 μM) versus absorbance with and without dithiotreitol. Reaction timewas 5 minutes;

FIG. 11 depicts a correlation between the quantitation results obtainedusing the CBS/CBL/LDH enzyme cycling system to measure homocysteine inhuman plasma samples using DTT as the reducing agent compared withquantitation results obtained by the Abbott IMx homocysteine method;

FIG. 12 depicts a correlation graph illustrating the correlation betweenthe assay of the present invention (y) versus the commercially availableIMx assay (x), as described in Example 7;

FIG. 13 depicts a correlation graph illustrating the correlation betweenthe assay of the present invention (y) versus a conventional HPLC assay(x), as described in Example 7;

FIG. 14 depicts a correlation graph illustrating the correlation betweenthe IMx assay (x) and the HPLC assay (y), as described in Example 7;

FIG. 15 depicts a graph of absorbance versus time for the no-DTTpyruvate assay described in Example 9; and

FIG. 16 depicts a graph of absorbance versus time for the DTT-containingpyruvate assay described in Example 9.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides a homogeneous enzymatic cycling assay forassessing the amount of homocysteine in a sample. Enzymatic cyclingmaintains a steady-state level of homocysteine and further provides anincrease in the sensitivity of assaying homocysteine. Further, thepresent invention provides genes and expression vectors for therecombinant production of enzymes that are useful in the presentinvention. Test kits for assessing the amount of homocysteine and/orcystathionine in a solution are also provided.

Homocysteine is present in cells as an intermediary amino acid producedwhen methionine is metabolized to cysteine. Generally, homocysteineproduced in the body is rapidly metabolized by one of two routes: (1)condensation with serine to form cystathionine or (2) conversion tomethionine, and its concentration (and that of its oxidized form,homocystine) in the body under normal conditions is low. The averageplasma homocysteine level for a healthy adult is about 12 μM for malesranging from about 8.6 to about 17.1 μM and 10 μM for females rangingfrom about 3.9 to 16.8 μM (Vester et al., Eur. J. Clin. Chem. Biochem.,29:549-554 (1991); Jacobsen et al. Clin. Chem., 40:873-881 (1994)).

Recently it has been determined that the amount of homocysteine levelsin a biological sample is of clinical significance in a number ofconditions. Elevated amounts of homocysteine in the blood have beencorrelated with the development of atherosclerosis (Clarke et al., NewEng. J. Med., 324:1149-1155 (1991)). Also, moderate homocysteinemia isnow regarded as a risk factor for cardiac and vascular disease. Accuracyin the normal range of homocysteine found in normal adults is veryimportant since the risk for heart disease has been determined toincrease markedly with as little as 30% over the normal concentrationincreasing the associated risk factor by 3.4 fold (Stampfer et al., JAMA268:877-881 (1992)).

As used herein the term “assessing” is intended to include bothquantitative and qualitative determination of the amount of homocysteineand/or cystathionine present in a test solution. A “solution” or “testsolution” as used herein refers to a clinical or biological fluid sampleor tissue extract and can include, for example, blood, bloodderivatives, such as plasma, or urine, and the like.

A sample containing homocysteine reacts with L-serine to formcystathionine in the presence of the enzyme CBS. Cystathionine can thenbe converted to homocysteine, pyruvate, and ammonia with a secondenzyme, CBL. The result of using these anabolic and catabolic enzymesoperating simultaneously is a “futile cycle,” whereby (1) cystathionineand homocysteine are continuously interconverted, (2) serine isdegraded, and (3) pyruvate and ammonia are created.

Use of relatively high concentrations of serine compared to homocysteineprovides for conditions conducive to an overall reaction rate whichfollows pseudo-first order kinetics dependent directly upon the amountof homocysteine and cystathionine in the reaction. If the concentrationof cystathionine is very low compared to the concentration ofhomocysteine, the rate of the overall reaction will vary linearly withthe amount of homocysteine. By measuring the reduction in serine or theincrease in either pyruvate or ammonia, the amount of homocysteine in anunknown solution or sample can be determined by a comparison withcontrol reactions comprising known concentrations of homocysteineundergoing the identical enzyme reactions. The enzymatic cycling methodof the present invention provides an amplification process producing fargreater amounts of end products than the amount of homocysteine likelyto be in a test solution, i.e., blood. Therefore, the assays todetermine the amount of pyruvate or ammonia produced by the reaction donot have to be extremely sensitive and can be measured by existingprocesses known to the skilled artisan.

Pyruvate is typically measured by the enzymatic conversion into lactatewith lactate dehydrogenase, or a derivative thereof. This enzymaticconversion reaction requires the cofactor NADH (reduced nicotinamidecofactor), or a derivative thereof, which is converted to NAD+ (oxidizednicotinamide cofactor). The reaction can be monitored by measuring theabsorbance at 340 nm. As used herein, the term “derivatives,” withrespect to cofactors, refers to salts, solvates, and other chemicallymodified forms that retain overall cofactor activity, but can haveextended stability in aqueous solutions. Derivatives can include, butare not limited to acetyl derivatives of NADH, including3-pyridinealdehyde-NADH, 3-acetylpyridine-NADH, 3-thioicotinamide-NADH,and the like (See, for example, U.S. Pat. No. 5,801,006).

Other convenient assays to determine the oxidation of NADH, or aderivative thereof, include for example, the measurement offluorescence, as described by Passoneau et al., in Enzymatic Analysis, APractical Guide, pages 219-222 (1993), (incorporated herein byreference). In another embodiment, NADH, or a derivative thereof, reactswith pyruvate in the presence of lactate dehydrogenase to form NAD+,which further reacts with a dye capable of producing a color change,preferably in the visible range, when oxidized. The color change of thedye can be used to determine the total amount of oxidation of NADH toNAD+ which corresponds to the amount of pyruvate formed from theenzymatic cycling reaction. Examples of dyes which can be used in thepresent invention include, but are not limited to,5,5′-dithiobis(2-nitrobenzoic acid), 2,6-dichlorophenolindophenol,tetrazolium compounds, phenazine methosulfate, methyl viologen, andderivatives of each.

Pyruvate can also be measured in a reaction with pyruvate oxidase, or aderivative thereof, in the presence of a peroxidase, i.e., horseradishperoxidase, and the like. The amount of pyruvate converted to hydrogenperoxide is determined by the oxidative condensation of, for example, awater soluble hydrogen donor which provides a colored compound forphotometric determination of the concentration of peroxide. Particularlypreferred hydrogen donors which provide a stable color reaction productinclude water soluble derivatives of N-alkyl-N-sulfopropylanilinederivatives, i.e., the sodium salt ofN-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS) (Tamaoku et al.,Chem. Pharm. Bull. 30:2492-2497 (1982)). The quantity of H2O2 can alsobe measured electrometrically. This includes amperometric andpotentiometric measurements. The determination of the amount ofhomocysteine and/or cystathionine is determined from the correlation ofthe amount of peroxide produced from the conversion of pyruvate bypyruvate oxidase over a defined time period.

Ammonia also can be measured by different methods, including by use ofan ammonia sensor (Willems et al., Clin. Chem. 34:2372 (1988). Ammoniacan also be readily detected using known colorimetric techniques. Forexample, ammonia generated in the sample can be reacted to form acolored product, the formation of which can be detectedspectrophotometrically. One such method, described in Methods ofEnzymatic Analysis (Bergmeyer) 1:1049-1056 (1970), incorporated hereinby reference, relies upon the reaction of ammonia with phenol in thepresence of hypochlorite in alkaline conditions to form the colored dyeindophenol. Sodium nitroprusside can be used as a catalyst.Modifications of the method using for example various derivatives canalso be used. The colored end product is formed in amounts directlyproportional to the concentration of ammonia, and hence homocysteine.Other methods can include, for example, microdiffusion analysis(Seligson et al., J. Lab. Clin. Med., 49:962-974 (1957), an ion exchangetechnique (Forman, Clin. Chem., 10:497-508 (1964), and various enzymaticmethods (See, for example, Mondzac et al., J. Lab. Clin. Med.,66:526-531 (1979)).

The enzymatic cycling method for assessing the amount of homocysteineand/or cystathionine reduces the difficulties seen in other prior assayswhere background compounds and background reactions produced “noise.” Inthe assay of the present invention, large amounts of an inexpensiveamino acid, e.g., L-serine, can be added to provide for the overallconversion of L-serine to pyruvate and ammonia. In a preferredembodiment, a serine concentration of 250 μM or more can be used, andsubsequently converted to pyruvate and ammonia. Plasma, typicallycontains about 100 μM serine. This amount of endogenous serine isimmaterial to the present assay since the assay measures a rate ofconversion that is limited by the amount of homocysteine and not theamount of serine. L-serine concentrations of about 0.1 mM to 10 mM wouldnot be expected to affect the sensitivity or accuracy of the disclosedassay.

Further, the enzymatic cycling reaction of the present inventionprovides a rate of L-serine conversion that is dependent upon theconcentration of homocysteine in a pseudo-first order reaction. Normalblood pyruvate levels are typically between about 0.4 and 2.5 mg/100 ml(Lehninger, A L. Principles of Biochemistry. 1982. WorthingtonPublishers, Inc. p. 707, incorporated herein by reference), whichcorresponds to about 45-284 μM. This concentration of pyruvate couldprovide a minor background measurement, but the present assay measures achange over time and can be easily adjusted for the starting level ofpyruvate in a solution. In addition, pyruvate may be removed by priorincubation with lactate dehydrogenase or other enzymes. Moreimportantly, cysteine and other sulfur-bearing compounds in, forexample, blood, do not contribute significantly to the enzymatic cyclingreaction and, therefore, are considered insignificant noise in thesystem.

In an alternative embodiment of the present invention the sample can betreated to remove cystathionine to increase the accuracy of thehomocysteine measurement. As a particular example, treatment comprisescontacting cystathionine γ-lyase with the solution to convertcystathionine to α-ketoglutarate (a compound which can not participatein the enzymatic cycling described above). Prior to proceeding with theenzyme cycling assay to assess the amount of homocysteine, thecystathionine γ-lyase activity must be removed from the sample ordestroyed, i.e., by heat, or the like.

This particular embodiment of the invention can also be used for theassessment of the amount of cystathionine by comparing the differencesin the rate of production of pyruvate and/or ammonia in a sample treatedand untreated with cystathionine γ-lyase prior to undergoing theenzymatic cycling assay. Therefore, the present invention can be used todetermine the amount of cystathionine in an unknown sample, even if thesample contains homocysteine.

In certain biological samples, such as blood, homocysteine is oftenbound through a disulfide linkage to proteins, thiols, and othercellular components. Typically, the sample can be treated with areducing agent, such as sodium or potassium borohydride; a thiol, suchas dithioerythritol, β-mercaptoethanol, dithiothreitol, thioglycolicacid or glutathione; or a phosphine or a trialkylphosphine, such astributylphosphine and tris(2-carboxyethyl)phosphine (TCEP), and thelike, to liberate bound and disulfide linked homocysteine. Prior assayswhich require antibodies for the detection step must change the redoxconditions of the reaction mixture (most antibodies are held togetherwith a disulfide bond). However, in the present method it is notnecessary to wash or alter redox conditions because the CBS and CBL usedare active under reducing conditions. Therefore, fewer steps arerequired for the assays of the present invention than for many prior artimmunoassays.

In a typical embodiment, a sample comprising blood, blood fractions,serum, plasma, or urine will be treated with a reducing agent anddirectly assayed for homocysteine. Heating of the sample prior to theassay to speed up the liberation of homocysteine and to inactivatedegradative enzymes and other factors that may interfere with the assaymay also be preferred. The general handling of blood samples forhomocysteine assays is known to those skilled in the art for othermethods such as HPLC tests.

CBS and CBL are enzymes commonly found in nature. Humans and yeast useCBS for the synthesis of methionine; and the human cDNA for CBS cansubstitute for the CBS gene in brewer's yeast (Kruger et al., Hum.Molec. Genet., 4:1155-1161 (1995)). CBL is found in many bacteria,including E. coli (Laber et al., FEBS Lett., 379:94-96 (1996). Theentire DNA sequences for the CBS and CBL genes are known in theseorganisms and in other species, providing a skilled molecular biologistwith a source to easily clone or synthesize these genes, or derivativesthereof.

As used herein “derivative” with respect to enzymes refers to mutantsproduced by amino acid addition, deletion, replacement, and/ormodification; mutants produced by recombinant and/or DNA shuffling; andsalts, solvates, and other chemically modified forms of the enzyme thatretain enzymatic activity of the isolated native enzyme. A method forcreating a derivative enzyme by DNA shuffling useful in the presentinvention is described in U.S. Pat. No. 5,605,793 (incorporated hereinby reference). Even without cloning, the two enzymes have been purifiedfrom a number of different organisms. See, for example, Ono et al.,Yeast 10:333-339 (1994); Dwivedi et al., Biochemistry 21:3064-3069(1982), incorporated herein by reference. In a preferred embodiment ofthe present invention genetically modified organisms are provided toproduce a CBS, or a derivative thereof, and to produce a CBL, or aderivative thereof, which can be used in the enzymatic cycling assay toprovide reagents at a reduced cost and to increase the ease of purifyingthe enzymes.

Similarly, the term “derivative” with respect to nucleotide sequencesrefers to variants and mutants produced by nuclueotide addition,deletion, replacement, and/or modification, and/or combinatorialchemistry; mutants produced by recombinant and/or DNA shuffling; andsalts, solvates, and other chemically modified forms of the sequencethat retains the desired activity of the sequence such as the bindingcharacteristics of MetR. Preferably, when modified derivative sequencesare used, the sequences have at least about 60% sequence identity withthe original sequence. More preferably, this identity is at least about70% and more preferably at least about 85%. Most preferably, suchsequences have at least about 95% sequence identity with the originalsequence.

The CBS gene and the entire genome of Saccharomyces cerevisiae have beensequenced. Likewise the CBL gene and the remaining genes of E. coli arealso known. Therefore, using techniques common to the art of molecularbiology, the genes encoding CBS and CBL can be cloned and expressed inboth organisms. Published methods for purifying the two enzymes areknown which use standard protein purification methods. However each ofthe enzymes can be constructed as fusion proteins including for example,a portion of another protein which assists in solubilization and/orrefolding of the active enzyme or amino acid sequences which can beadded to aid in purification. An example of amino acid sequences addedfor purification include, affinity tags, such as poly-His, orglutathione-S-transferase (GST) can be added to allow for the more rapidpurification of the proteins over a single affinity column. Although theenzymes do not require purification to homogeneity for a diagnostic testof the present invention, proteases and other activities that convertserine to pyruvate can be reduced to insignificant levels bypurification.

In addition, concentration of the enzymes generally leads to higherstability of enzyme activity during the reaction and upon long termstorage. CBS and CBL from organisms other than S. cerevisiae and E.coli, especially thermophiles, are expected to be sources of enzymeshaving even greater stability. The isolated enzymes can also be selectedfor a higher affinity to homocysteine. In addition, protein engineeringof the yeast CBS and bacterial CBL can lead to enzymes with improvedcommercial properties, including higher affinities, faster reactionrates, easier purification, and a longer half-life upon storage.

Similarly, cystathionine γ-lyase (CGL) has been described in detail inmany species (see, for example, Yamagata, et al., J. Bacteriol.,175:4800-4808 (1993) incorporated herein by reference). Manipulation ofthe genes which encode CGL can be further modified genetically bystandard recombinant methods and inserted into a host cell to develophigher level production strains.

Recombinant expression of the genes for CBS, CBL, and CGL are typical.These methods can provide large quantities of purified enzyme and thegenes can be modified, for example, to add a defined peptide sequence(an affinity tag) to the expressed enzymes that can be used for affinitypurification. Through the use of affinity tags in the expression of therecombinant enzymes, the purification and immobilization of theseenzymes can be greatly simplified, leading to lower costs. Affinity tagsare known in the art and include for example, but not limited,poly-histidine, GST (glutathione-S-transferase), strep-tag, “flag,”c-myc sequences, and the like. Tags are widely used for affinitypurification of recombinantly expressed proteins, and many arecommercially available.

In a preferred embodiment, CBS and CBL are immobilized on a solidsurface in a manner that maintains the activities of the enzymes and ina manner that keeps homocysteine free to interact directly with theproteins. By immobilizing the enzymes at high densities, the diffusiondistances between the different proteins are reduced relative to enzymesin solution. As a result, the overall rate of reaction of the assayincreases greatly, since the product of one enzyme can be reacted uponby the other enzyme and vice versa. Acceleration of the reaction ratesis due to a local increase in the concentrations of the intermediates,which are in closer proximity to the next enzyme as a result ofimmobilization. Increasing the rate of the homocysteine assay can be animportant factor for the marketability of the diagnostics productbecause the K_(m) of CBS and of CBL are in the millimolar range but theblood concentrations of homocysteine are approximately 10 μM.Diffusion-limited reactions are known to require a high concentration ofthe enzymes to provide a rapid reaction rate. Immobilization of theenzymes in the cycling assay overcomes difficulties which might bepresented by the low affinities of these two enzymes for theirrespective substrates.

In yet another preferred embodiment CBS and CBL are immobilized as afusion protein. The distance and orientation of the two differentproteins relative to each other can be controlled during theconstruction of the fusion gene expression vector. By properlypositioning the two activities in a fusion protein, homocysteine andcystathionine interconversion can occur extremely rapidly virtuallywithin the protein, since diffusion distances can be minimized betweenthe two active centers. The homocysteine, therefore, behaves like acofactor in the overall enzymatic cycling reaction, which makes pyruvateand ammonia from L-serine.

The enzymatic cycling assay of the present invention can be provided ina number of assay formats. The simplest format of the assay provides areagent solution comprising L-serine, CBS, or a derivative thereof, CBL,or a derivative thereof, and various buffers and auxiliary substancesnecessary for optimization of the enzyme reactions. In a particularlypreferred embodiment of the present invention CBS, or a derivativethereof, and CBL, or a derivative thereof, are provided immobilized on asolid surface.

Immobilization of enzymes to a solid surface while retainingsubstantially all the activity of the enzyme are well known in the art.Several methods have been used for immobilization including, covalentbinding, physical absorption, entrapment, and electrochemicaldeposition. As one example glutaraldehyde has been used to immobilizecreatinine deaminase and glutamate oxidase to propylamine derivatizedcontrolled pore glass beads (Rui et al., Ann. NY Acad. Sci., 672:264-271(1992)). Other examples include, but are not limited to, the use ofcarbodiimides to covalently attach enzymes to succinate derivatizedglass beads (Rui et al., supra), and the use of hetero- orhomo-bifunctional cross-linking reagents (e.g., SPDP, N-succinimidyl3-(2-pyridyldithio)propionate; SMPT,succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene; DSP,dithiobis(succinimidylpropionate); DTSSP,3,3′-dithiobis(sulfosuccinimidylpropionate); DSS, disuccinimidylsuberate, and the like) to covalently attach an enzyme through, forexample an amine or sulfhydryl group to a derivatized solid surface(Wilson, et al., Biosens. Bioelectro., 11:805-810 (1996)).

The “solid surface” as used herein can include a porous or non-porouswater insoluble material that can have any one of a number of shapes,such as a strip, rod particle, including a bead or the like. Suitablematerials are well known in the art and can include, for example, paper,filter paper, nylon, glass, ceramic, silica, alumina, diatomaceousearth, cellulose, polymethacrylate, polypropylene, polystyrene,poly-ethylene, polyvinylchloride, and derivatives of each thereof. Thesolid surface can be the sides and bottom of the test container or canbe a material added to the test container. In one preferred embodimentthe solid surface comprises a bead which is added to the test container.

Both yeast and bacteria have enzymes that convert L-serine to pyruvate,activities that are sometimes referred to as serine deaminase or serinedehydratase. Well established mutagenic methods can be used to greatlyreduce or eliminate these activities, which can contributedsignificantly to the background in the homocysteine cycling assay.Saccharomyces cerevisiae, in particular, may be genetically manipulatedto delete out the serine deaminase activities, which are necessary for(1) isoleucine synthesis and (2) growth on serine as a carbon ornitrogen source, by using a simple method known as “gene disruption.” Bydeleting the chal gene of yeast, a major serine deaminase activity isremoved, thereby eliminating one potential background problem to thecycling assay. In addition, the cha1 deletion can be useful forselecting CBS-CBL fusion proteins and for genetically engineering thosefusion proteins. The cha1 deletion strain is unable to grow on serine asthe sole carbon or nitrogen source. The CBS-CBL fusion gene is able tocomplement the deletion, so long as homocysteine is available as a“cofactor,” because the cycling system produces the same net result asthe serine deaminase. Therefore, the cha1 deletion can be used to selectfor and maintain improved CBS-CBL fusions, especially if a population ofthe fusion genes is mutated and placed on single-copy vectors or ontochromosomes in yeast. Cells that grow faster on serine media or have areduced requirement for homocysteine may contain mutations that code forfaster enzymes or proteins with higher affinities for homocysteine andcystathionine.

The present invention also provides, as an alternative method forassessing the amount of homocysteine in a sample, the use of ametabolite binding transcription factor. When a metabolite bindingtranscription factor is bound to their respective metabolite the factorcomplex binds to a specific receptor polynucleotide sequence. Thebinding of the transcription factor complex to its receptor consensussequence can be monitored and correlated to the amount of the metabolitepresent in the sample.

A number of transcription factors that bind to specific DNA bindingsites upon association with a small molecule or metabolite are known inthe art. For example, E. coli has an operon regulatory element, thetranscription factor MetR which regulates the Met operon in response tobinding to homocysteine (Cai et al., Biochem. Biophys. Res. Commun.,163:79-83 (1989)). MetR or derivatives thereof regulate genes such asMetE (cobolamin independent methionine synthase), MetH (cobolamindependent methionine synthase) and GlyA (serine hydrogenase). MetR andits derivatives preferentially bind to its consensus DNA sequence in thepresence of homocysteine and is free in the cell cytoplasm in itsabsence. The binding of MetR in the presence of homocysteine can beexploited to assess the amount of homocysteine in a sample suspected ofcontaining homocysteine.

In a particular embodiment of the invention the assay comprises apolynucleotide sequence encoding the consensus sequence for a receptorpolynucleotide for MetR immobilized on a solid support in a manner whichallows for the accessibility of the consensus receptor sequence forbinding to MetR. The nucleotide sequence can be, for example:

-   GTTAATGTTGAACAAATCTCATGTTGCGTG (SEQ ID NO: 11)

A sample, such as plasma, blood or urine, is treated to release anyprotein bound homocysteine prior to admixing the sample with a reagentsolution comprising MetR in a buffering agent in the presence of theimmobilized consensus receptor sequence under conditions conducive tocomplex formation. After an incubation period the solid support iswashed to remove unbound reagents and the amount of MetR that remainsbound to the solid support is assessed. The determination of the amountof MetR that remains bound to the solid surface typically can be anELISA, surface plasmon resonance, i.e., BIACORE, or simply a standardprotein assay, and this amount is correlated with the amount ofhomocysteine present in the sample.

Treatment of the sample to release homocysteine can be accomplished witha reducing agent. As described above a number of agents are well knownin the art. Typically a sample will be admixed with a buffer solutioncontaining a sufficient amount of reducing agent to releasesubstantially all of the homocysteine from any associated proteinspresent in the sample. Commonly used reducing agents include, but arenot limited to, sodium and potassium borohydride, β-mercaptoethanol,dithiothreitol, dithioerythritol, thioglycolic acid or glutathione; or aphosphine or a trialkylphosphine, such as tributylphosphine andtris(2-carboxyethyl)phosphine (TCEP), and the like.

The MetR protein can also be modified to incorporate a detectable label,such as a chromophore, a fluorophore, and the like, to improve the speedand sensitivity of the assay. The addition of the detectable label canreduce the time period required to run the assay by eliminating certainsample handling steps, for example, additional washing steps.Incorporation of a fluorophore that emits light at the wavelengthabsorbed by oligonucleotides into the consensus binding sequence allowsfor the measurement of fluorescent energy transfer to quantify thebinding of the protein to the consensus sequence. Methods which can beused to measure fluoresence transfer include, for example, Fluorescenceresonance energy transfer (FRET) or a proximity scintillation assay.Additional modification of the MetR binding consensus sequence by theaddition of an additional detectable label within the binding domain canalso improve the specificity of the assay.

Additionally, a defined peptide sequence (an affinity tag) can be addedto the MetR protein to aid in purification and detection. Affinity tagsare known in the art and include for example, but not limited,poly-histidine, strep-tag, “flag,” c-myc sequences, and the like. Tagsare widely used for affinity purification of recombinantly expressedproteins, and many are commercially available. Further, mutationalalterations to improve binding affinity of a recombinantly expressedMetR can be used to provide additional assay reagents.

In a separate embodiment of the present invention, the amount ofhomocysteine in a sample can be determined by the enzymatic utilizationof homocysteine in the sample. Conversion of homocysteine in cellularmetabolism typically releases a proton from the sulfhydryl residue ofhomocysteine. Release of this proton was used to measure the bindingaffinity of homocysteine to E. coli cobolamin dependent methioninesynthase (Jerret et al., Biochemistry, 36:15739-15748 (1997)). Enzymereactions catalyzed by proteins, such as methionine synthase, generallyrequire cofactors or additional substrates to complete the reaction. Ifthese cofactors and substrates are not present, the homocysteine remainsbound to the enzyme. In the present method, measurement of the releasedproton removes the proton released by the enzymatic reaction driving thereaction toward consumption of the homocysteine present in the sample.The measurement of the change in pH associated with the released protonsprovides a determination of the amount of homocysteine in the sample. Ina preferred embodiment, for example, cobolamin independent methioninesynthase, can be used in the assay.

As a matter of convenience, the reagents for use in the presentinvention can be provided in a test kit for use in the assay method. Atypical kit comprises a packaged combination of a container to hold thetest solution, L-serine, CBS, or a derivative thereof, and CBL, or aderivative thereof, and auxiliary buffers and substances required foroptimal reaction conditions. Importantly, a variety of preservativesand/or stabilizing agents may also be included with any/all kit reagentsand/or enzymes to elongate shelf life as well as “on-line” life forsample testing. The kit can further comprise reducing agents or CGL forpretreating the sample prior to the cycling assay. Further, the kit caninclude lactate dehydrogenase, NADH and a dye capable of an observablecolor change upon oxidation.

Under appropriate circumstances one or more of the reagents in the kitcan be provided in solution (with or without preservatives and/orstabilizing agents) or as a dry powder, usually lyophilized, includingexcipients, preservatives, and/or stabilizing agents, which ondissolution can provide for a reagent solution having the appropriateconcentrations for performing the assay in accordance with the presentinvention. To enhance the versatility of the subject invention, thereagents can be provided in packaged combination, in the same orseparate containers, so that the ratio of the reagents provides forsubstantial optimization of the method and assay. The reagents can eachbe in separate containers or various reagents can be combined in one ormore containers depending on the stability of the reagents. As a matterof convenience, the reagents employed in the assay method of the presentinvention can be provided in predetermined amounts. The test kit canalso contain written instructions on how to use the reagents and/or howto perform a particular assay, for example in the form of a packageinsert.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE 1 Cloning and Expression of the E. coli Cystathionine β-LyaseGene

In this example standard PCR procedures were used to clone the E. coliCBL gene. The isolated gene was inserted into an expression vector withan affinity tag sequence (6HIS), and the expressed protein was purifiedwith an affinity column. A substantial amount of the CBL enzyme activityin the host cells transformed with the expression vector construct waspurified from the cell lysates.

Cloning of the E. coli Cystathionine β-lyase (CBL, EC 4.4.1.8) Gene

Standard PCR procedures using EXPAND HIGH FIDELITY PCR system (RocheBiochemicals) were employed to amplify the E. coli CBL gene using ONESHOT TOP10 competent E. coli cells (Invitrogen) as the genomic DNAsource. PCR primer sequences for CBL cloning were as follows: (CBLN-terminal primer: 5′-CGACGGATCCGATGGCGGACAAAAAGCTTG-3′; SEQ ID NO: 1)and (CBL C-terminal primer: 5′-CGCAGCAGCTGTTATACAATTCGCGCAAA-3′; SEQ IDNO: 2). The PCR amplified CBL gene product was then purified by agarosegel electrophoresis, digested with BamHI and PvuII restrictionendonucleases, and purified again by agarose gel electrophoresis. Thepurified E. coli CBL gene was then ligated into gel purified,BamHI/PvuII digested pRSETB protein expression vector (Invitrogen). Theresulting construct provided the CBL gene under control of the T7transcriptional promoter, cloned in frame with an amino terminal sixHistidine (6HIS) residue leader sequence (hereafter referred to as6HISCBL) which allows for convenient expression, affinity purification,and immunostaining analysis of recombinant 6HISCBL protein from E. colicells. The DNA sequence of E. coli Cystathionine β-lyase (CBL) gene ispresented (SEQ ID: 21). The protein sequence of the cloned E. coliCystathionine β-lyase (CBL) having an amino-(NH3)-terminal 6Histidine(6His) affinity tag, resulting from cloning the CBL gene into the pRSETBbacterial expression plasmid is also presented (SEQ ID: 19).

Cystathionine β-Lyase Protein Expression and Purification

The pRSETB(6HISCBL) plasmid construct was transformed into BL21(DE3)pLysS E. coli strain (Invitrogen), spread on LB/Agar platescontaining (100 μg/ml ampicillin, 35 μg/ml chloramphenicol), and wasthen incubated overnight at 37° C. Single bacterial colonies were thentransferred and grown overnight in flasks containing 100 ml LB growthmedium containing ampicillin and chloramphenicol. The 100 ml overnightculture was then added to multiple flasks containing 1 L of fresh LBgrowth medium containing ampicillin, and allowed to grow until reachingan A₆₀₀ of 0.6 (approx. 2.5 hours). In order to induce CBL proteinexpression (driven by the T7 promoter),isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to the growthmedium to a 0.1 to 1 mM final concentration. The CBL enzyme cofactorpyridoxal 5′-phosphate (PLP) was also added to a final concentration of100 μM in the growth medium during induction. After monitoring cellgrowth and 6HISCBL protein expression over a 5 hour period, E. colicells were harvested by centrifugation, resuspended, and lysed by theaddition of BUGBUSTER Protein Extraction Reagent (Novagen Inc.) in thepresence of EDTA free complete protease inhibitor (Roche Biochemicals)as described in the manufacture's protocol. The cell lysate was thentreated with DNase I, cleared by centrifugation and loaded onto anaffinity column containing Poly-His Protein Purification Resin (RocheBiochemicals). The column was then washed with 10 bed volumes ofequilibrating buffer (50 mM KPi, pH 7.8, 0.5 M NaCl), followed byelution with 5 bed volumes of elution buffer 1 (50 mM KPi, pH 7.8, 0.5 MNaCl, 10 mM imidazole; EB1), 5 bed volumes of elution buffer 2 (50 mMKPi, pH 7.8, 0.5 M NaCl, 50 mM imidazole; EB2), and 5 bed volumes ofelution buffer 3 (50 mM KPi, pH 7.8, 0.5 M NaCl, 500 mM imidazole; EB3).Samples taken during each step of the purification procedure were thenanalyzed by denaturing protein gel electrophoresis (SDS-PAGE) andstained with either Coomassie Brilliant Blue, or subjected to Westernblot analysis using a monoclonal anti-polyhistidine peroxidaseconjugated mouse immunoglobulin for detection as described by themanufacturer (Sigma).

The purified 6HISCBL protein was found to elute primarily in elutionbuffers 2 and 3 (EB2 and EB3). The CBL containing eluate was thenconcentrated using centrifugal filtration chambers (Amicon) and dialyzedovernight at 4° C. against storage buffer (50 mM Tris HCl, pH 8, 100 mMNaCl, 5 mM PLP), aliquoted and stored frozen at −80° C. Theconcentration of purified homogenous CBL enzyme was then calculated onthe basis of the extinction coefficient at 280 nm and a subunit weightof 43,312 Da. (Clausen et al. Biochem. 36:12633-42 (1997)). A typicalyield of purified 6HISCBL was in the range of 20-30 mg 6HISCBL per gramof E. coli cell paste.

CBL Activity Assay

The CBL activity of affinity purified recombinant 6HisCBL proteinexpressed from the pRSETB(6HisCBL) plasmid construct was performedessentially as described previously (Clausen et al., Biochem.36:12633-42 (1997) incorporated herein by reference). Briefly, assaymixtures contained 100 mM Tris/HCl (pH 8.5), 5 mM L-cystathionine, 1 mM5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent), and6HisCBL enzyme in a final reaction volume of 1 mL. The reaction wasfollowed by recording the increase in absorbance at 412 nm with time.The same enzyme reaction was also carried out on samples having knownconcentrations of substrate to produce a standard curve. Substantiallyall of the CBL activity detected in the whole cell lysate was recoveredand found present in the experimentally purified samples of recombinant6HisCBL enzyme produced from the pRSETB(6HisCBL) plasmid construct.

EXAMPLE 2 Cloning and Expression of the Yeast Cystathionine β-Synthase(CBS) Gene in Yeast

In this example PCR methods were used to clone the CBS gene from yeast.The PCR fragment comprising the CBS gene was inserted into an expressionvector both with and without an affinity tag which was added to assistin purification of the expressed gene product.

A laboratory strain of Saccharomyces cerevisiae (5153-11-1 (his3 met2))was used as the source of the CBS gene. The cells were grown on YEPD at32° C., spun down, washed with water, and transferred into 200 μl ofYeast Plasmid Buffer (100 mM NaCl, 10 mM Tris (pH 8), 1 mM EDTA, 0.2%SDS). The cell suspension was mixed with an equal volume of 0.45 mmglass beads and vortexed at high speed for 3×30 seconds. The liquid wasremoved by pipetting and centrifuged for two minutes in a microfuge toremove cell debris. The supernatant was extracted with PCI(phenol:chloroform:isoamyl alcohols (25:24:1) and chloroform. Sodiumchloride (5 M) was added (about 20 μl), and the DNA was precipitatedwith an equal volume of isopropanol.

This genomic yeast DNA was amplified by PCR with primers CI-001(CGGGGATCCTGATGCATGCATGATAAGGA; SEQ ID NO: 3) and CI-012(CGGCATTACCGTTTCACTAATTTATTG; SEQ ID NO: 4), which are publishednucleotide sequences that flank the structural gene for the yeast CBS.Primer CI-001 (SEQ ID: 3) contains a BamHI site found on theuntranslated 3′ side of the CBS gene. To add a BamHI sequence to the 5′side of the PCR fragment, the gene was further amplified by PCR withCI-001 (SEQ ID NO: 3) and CI-002 (GTTGGATCCGGCATTACCGTTTCAC; SEQ ID NO:5). The resulting PCR product was a DNA sequence of approximately 1937base pairs, including 1521 base pairs that encode the CBS protein. Theadditional DNA that flanks the coding sequence comprises the naturalpromoter and transcription termination sequences of the yeast CBS gene.

A stronger promoter was inserted upstream of the CBS gene to promotehigher levels of protein expression. The yeast TPI1 promoter was clonedfrom the same genomic yeast DNA using the primers CI-009(GGTGGATCCATCAATAGATACGTACGTCCT; SEQ ID NO: 6) and CI-010(TTTTAGTTTATGTATGTGTTTTTTG; SEQ ID NO: 7) in a standard PCR reaction. Toattach the CBS coding region to the TPI1 promoter, an adapter primer isused: CI-011 (AACACATACATAAACTAAAAATGACTAAATCTGAGCAGCAA; SEQ ID NO: 8),which contains 20 bases of the TPI1 promoter and the first 21 bases ofthe CBS gene. The CBS sequence obtained above was amplified by PCR withprimers CI-001 (SEQ ID NO: 3) and CI-011 (SEQ ID NO: 8). The resultingPCR product was mixed with the TPI1 promoter product from above, and themixture was amplified by PCR with primers CI-001 (SEQ ID NO: 3) andCI-009 (SEQ ID NO: 6) to produce a fusion gene designated “TPICBS,” thatcomprising the TPI1 promoter, the CBS coding sequence, and the CBStranscription terminator.

The CBS and TPICBS PCR products each comprise BamHI sites on both the 5′and 3′ sides of the coding region. These PCR products were purified witha PCR WIZARD PREP (Promega), according to the manufacturer'srecommendations, and were cut with BamHI. The resulting BamHI nucleotidefragments were run on a 1% Agarose TBE Gel and purified with NA45 paper(S&S). The yeast, E. coli shuttle vector C1-1 has a unique BamHI site inthe gene for tetracycline resistance was used for expression of the CBSgene. C1-1 vector was cut with BamHI and treated with shrimp alkalinephosphatase, according to the manufacturer's instructions (RocheBiochemicals). The CBS and TPICBS BamHI fragments were ligated into thelinearized C1-1 vector. The ligation reaction was transformed into TOP10cells (Invitrogen), which were plated onto LB ampicillin plates.Amp-resistant transformants were transferred onto LB amp and LB tetplates to determine tetracycline sensitive clones. Several tet-sensitivecolonies were selected from the LB amp plates, grown overnight on LB ampplates, and were further processed by the alkaline lysis method forplasmid DNA. The DNA was cut with BamHI and separated on a 1% agarosegel to determine which plasmids contain the CBS or TPICBS inserts.

C1-1 plasmids that carry inserts were transformed into the yeast strain,INVSc-1, a diploid strain that is commercially available fromInvitrogen. INVSc-1 has a mutation in both copies of the LEU2 gene andrequires the LEU2 gene from the C1-1 plasmid for selective growth onleucineless media. INVSc-1 was transformed with the C1-1 relatedplasmids, using the Yeast Transformation Kit from Invitrogen, accordingto the manufacturer's instructions. This kit involved makingspheroplasts of the cells with Zymolyase, followed by a calciumtreatment for DNA transformation. The transformants were plated onto asynthetic medium containing 1M sorbitol, yeast nitrogen base, and yeastsupplements, excluding leucine. Transformants were visible in theoverlay agar after three days of incubation at 32° C.

Attachment of Poly-Histidine to Cystathionine β-Synthase for YeastExpression

DNA sequences that encode six histidines were added to the 3′ end of theCBS coding region, in both the CBS and TPICBS vector constructs.Poly-histidine coding sequences were added to the original CBS clone byamplification of the CBS PCR fragment from above with the primer CI-017(ATGATGATGATGATGATGACCTGCTAAGTAGCTCAGTAA; SEQ ID NO: 9) and primerCI-002 (SEQ ID NO: 5). The resulting PCR product comprises the naturalCBS promoter region, the CBS coding region, and a further codingsequence for glycine and six histidine residues. The glycine residue wasadded to increase the spatial flexibility of the poly-his peptide aftertranslation. The poly-his CBS PCR product was gel-purified away from theoriginal CBS DNA and was amplified by PCR sequentially twice thereafterwith primers, CI-002 (SEQ ID NO: 5) and CI-017 (SEQ ID NO: 9) tovirtually eliminate the original CBS DNA.

Similarly, the TPICBS PCR product from above was amplified with primers,CI-009 (SEQ ID NO: 6) and CI-017 (SEQ ID NO: 9). The PCR product was gelpurified and reamplified twice with CI-009 (SEQ ID NO: 6) and CI-017(SEQ ID NO: 9) to greatly reduce the input TPICBS DNA sequence.

The CBS gene was also cloned by PCR with primers CI-001 (SEQ ID NO: 3)and CI-018 (CATCATCATCATCATCATTAAATAAGAACCCACGCT; SEQ ID NO: 10) toproduce a poly-histidine sequence with a TAA stop codon, followed by thetranscription terminator sequence. This short PCR product (“terminator”)of about 190 bp was also gel purified and cloned by PCR with the sameprimers. The poly-his CBS and “terminator” fragments were combined intoone PCR reaction with the primers, CI-001 (SEQ ID NO: 3) and CI-002 (SEQID NO: 5). The resulting amplification produced a nucleic acid moleculedesignated “CBSH,” comprising the natural CBS promoter, the entire CBScoding region, one additional glycyl residue, six histadyl residues witha stop codon, and the CBS transcription terminator.

Likewise, the poly-his TPICBS and terminator fragments were combinedinto one PCR reaction with the primers, CI-001 (SEQ ID NO: 3) and CI-009(SEQ ID NO: 6). The resulting nucleic acid product, designated “TPICBSH”was the same as the CBSH sequence, except that the TPI1 promoterreplaced the natural CBS promoter. The CBSH and TPICBSH (each containinga poly-histidine affinity tag sequence) were cut with BamHI and ligatedinto C1-1, as done above for the CBS and TPICBS sequences. The ligationmixes were transformed into INVSc-1. Tet-sensitive colonies wereexamined for the insertion of the appropriate cloned genes into C1-1 byrestriction enzyme analysis.

EXAMPLE 3 Cloning, Expression, and Purification of the YeastCystathionine β-Synthase (CBS) Gene in E. coli

In this example standard PCR and molecular biology techniques were usedto clone the Saccharomyces cerevisiae (INVSc1) Cystathionine β-Synthase(CBS) gene as a fusion protein with an amino-terminal glutathioneS-transferase (GST). Addition of this region of glutathioneS-transferase allows for the single-step affinity purification of theCBS protein from the E. coli culture medium and may also assist insolubilization and/or to promote proper refolding of the enzyme in anactive form.

Cloning

The laboratory strain of Saccharomyces cerevisiae (INVSc1) (Invitrogen)was used as the source of CBS gene. A single colony of INVSc1 yeast wasboiled in 100 μl of deionized water for 5 min. and vortexed rapidly inthe presence of an equal volume of 0.45 mm glass beads for 2 min. Onemicroliter of this cell lysate was then used as the source of DNA forPCR amplification of yeast CBS gene using primers CI-024(GCGGGTCGACTATGACTAAATCTGAGCAGCAAGCC; SEQ ID NO: 12) and CI-025(GCGTGCGGCCGCGTTATGCTAAGTAGCTCAG; SEQ ID NO: 13) which contain flankingsequences to the published yeast CBS gene and restriction sites forcloning into the pGEX-6P-2 (Amersham Pharmacia) expression vector. Theresulting PCR product (approximately 1548 bp) was then isolated andpurified by agarose gel DNA extraction using a commercial kit (Roche).The purified PCR product containing the CBS gene flanked by SalI (5′end) and NotI (3′ end) restriction enzyme sites was then digested (bySalI and NotI), gel purified, and ligated into the pGEX6P-2 expressionvector (Amersham Pharmacia). Restriction enzyme mapping was used toconfirm the identity of the PCR amplified DNA as the yeast CBS gene. TheDNA sequence for the cloned Saccharomyces cerevisiae Cystathionineβ-Synthase (CBS) gene is depicted (SEQ ID: 14). The protein sequence ofthe cloned Saccharomyces cerevisiae CBS having an amino-(NH3)-terminalGST fusion protein attached as a result of cloning into the bacterialexpression vector pGEX6P-2 is also presented (SEQ ID: 20).

Expression and Purification

The pGEX6P-2 expression vector containing the cloned yeast CBS gene(described above) was transformed into TOP10 E. coli cells (Invitrogen).Single colonies were picked and cell cultures were grown in LB mediacontaining 100 μg/ml ampicillin at 30EC until reaching an absorbance at600 nm of approx. 0.5 OD. In order to induce expression of the GST-CBSfusion protein, (driven by the tac promoter),isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the growthmedium to a final concentration of 0.1 mM. The CBS cofactor, pyridoxal5′ phosphate, was also added to the growth medium at 100 μM finalconcentration during induction. Induced cells were then allowed to growat 30° C. for an additional 21 hrs before harvesting by centrifugation.

Affinity purification of the GST-CBS protein using Glutathione Sepharose4B affinity resin was then performed essentially as described in themanufacture's protocol (Amersham Pharmacia). Briefly, cells weresuspended in phosphate buffered saline (PBS) and disrupted by sonicationon ice. TritonX-100 was then added to a final concentration of 0.1%(v/v), and the sonicate was shaken at room temperature of 30-45 min. Thecell lysate was then cleared of insoluble debris by centrifugation,treated with DNase I, and loaded onto a column of Glutathione Sepharose4B affinity resin. Nonspecifically bound proteins were washed away with10 column volumes of PBS, and the desired GST-CBS protein was theneluted from the affinity resin by the addition of 5 column volumes ofelution buffer (50 mM Tris, pH 8, 1.4 mM β-mercaptoethanol (BME), 100 mMNaCl, 0.1% (v/v) TritonX-100, 50 mM reduced glutathione). Purificationof the GST-CBS fusion protein was then confirmed by Coomassie Bluestained SDS-PAGE (sodium dodecylsulfate, polyacrylamide gelelectrophoresis). The eluted GST-CBS was then dialyzed into storagebuffer (50 mM Tris HCl, pH8, 100 mM NaCl, 5 μM PLP), aliquoted andstored at −80° C. The concentration of purified GST-CBS was thendetermined using the MICRO BCA Protein Assay Kit (Pierce Chemical). Atypical yield of purified GST-CBS was in the range of 5-10 mg GST-CBSper gram of E. coli paste.

Activity Assay for CBS

The CBS activity of the purified yeast GST-CBS fusion protein wasperformed as described by Kashiwamata and Greenberg (Biochim. Biophy.Acta 212:488-500 (1970)). This assay is based on the ability of CBS tosynthesize cystathionine from serine and homocysteine. The amount ofcystathionine formed by CBS was then determined spectrophotometricallyby detecting the ninhydrin reaction chromagen at 455 nm and compared toa standard curve generated using samples of known cystathionineconcentrations. CBS activity was also determined by the enzymatic assaysdescribed infra, or by other assays well known to the skilled artisan.

EXAMPLE 4 Construction of ADH2CYS4 Fusion Construct

Expression and Purification of Yeast Cystathionine β-Synthase in Yeast

In this example a strong promoter, ADH2, was inserted upstream of theCYS4 gene and constructed as a fusion with the yeast CBS gene to promotehigher protein expression. The ADH2 promoter is regulated by the Adr1p,and is repressed when yeast cells are grown in glucose containing mediumand becomes derepressed when the cells are grown in a non-fermentablecarbon source. This allows for regulated expression, if overproductionof a protein is lethal to the cell.

Construction of ADH2CYS4 Gene

Yeast genomic DNA was amplified with primers CI-029(CTATATCGTAATAATGACTAAATCTGAGCAGCAAGCCGATTCA; SEQ ID NO: 15) and CI-017(ATGATGATGATGATGATGACCTCGTAAGTAGCTCAGTAA; SEQ ID NO: 9) to produce allof the CYS4 gene without the stop codon and transcription terminator.CI-029 (SEQ ID NO: 15) primer contains the last 13 bases of the ADH2promoter region cloned in addition to the first ten codons of CYS4 gene.Primer CI-017 (SEQ ID NO: 9) has the sequence for the last six codons ofCYS4 gene, except the stop codon plus a glycyl residue and six histadylresidues. The resulting PCR product comprises the CYS4 coding region,and a further coding sequence for glycine and six histidine residues.The glycine was added to increase the spatial flexibility of thepoly-his peptide after translation. The “terminator” fragment wasproduced by amplifying the same genomic DNA with primers CI-018(CATCATCATCATCATCATTAAATAAGAACCCACGCT; SEQ ID NO: 10) and CI-040(AGGGCTCGAGGATCCCGGGGGTGCTATTATGAATGCACGG; SEQ ID NO: 16) to produce apoly-histidine sequence with a TAA stop codon, followed by thetranscription terminator. The CI-029/CI-017 PCR reaction produced anapproximately 1530 bp fragment while CI-018/CI-040 reaction produced anapproximately 331 bp fragment. These two fragments were joined by mixingtheir PCR products and amplifying with primers CI-029/CI-040. Theresulting product was designated CYS4H and was approximately 1861 bp inlength.

To join the ADH2 promoter to the CYS4H gene fragment, a PCR product wasfirst generated with primer CI-027 (CGGGGATCCGACGTTTTGCCCGCAGGCGGGAAACC;SEQ ID NO: 17) and primer CI-028(AGATTTAGTCATTATTACGATATAGTTAATAGTTGATAG; SEQ ID NO: 18) from the sameyeast genomic DNA to produce the ADH2 promoter sequence. Primer CI-028(SEQ ID NO: 18) contains the first 12 nucleotides of the coding regionof CYS4 gene. Therefore, between primers CI-028 (SEQ ID NO: 18) andCI-029 (SEQ ID NO: 15) there are 25 nucleotides of complimentarity DNAin their respective products. The ADH2 PCR product was mixed with CYS4Hfragment and amplified with primers CI-027 (SEQ ID NO: 17) and CI-040(SEQ ID NO: 16), to produce a product fragment of approximately 2201 bpdesignated ADH2CYS4H. Each of these PCR products was confirmed byrestriction endonuclease analysis.

The ADH2CYS4H product was cloned into YEp24 by cutting both DNA withBamHI and ligating using T4 DNA ligase. Ligated products weretransformed into E. coli (TOP10; Invitrogen) cells and plated on LB-agarplus ampicillin plates. Plasmids, prepared using Plasmid Preparation kit(Roche) according to the manufacture's recommendation from putativerecombinants, were analyzed by restriction endonuclease. The plasmidsfrom clones found to have the correct insert were transformed into S.cerevisiae (BJ5460) and plated on synthetic complete medium minus uracil(SC-ura), supplemented with 2% glucose. After two days putativerecombinants were re-streaked on the same type of plates to isolatecolonies. The cells are induced by growing in SC-ura plus 2% ethanol and1% peptone, usually supplemented with 0.1% glucose to enable the cellsto grow within 24 hours. Following overnight growth cells were arecollected by centrifugation and the cell pellet was saved at −70° C.until lysis. Cells were lysed by resuspending the cell pellet in 0.05volume of the original volume in lysis buffer. Glass beads were thenadded to the meniscus of the buffer.

The whole suspension was vortex for 1 min each for four times. Cellswere cooled on ice for 1 min between vortexing. Lysate was transferredto fresh tube and centrifuged for 10 min at 10,000×g. The cleared lysatewas layered onto a His-Resin column (Roche) and the protein was purifiedaccording to manufacture's recommendation. A partially purified proteinwas recovered in elution three.

EXAMPLE 5 Examples of Non-Cycling Portion of Assay Formats forHomocysteine

In this example the non-cycling portion of the assay for pyruvate andcystathionine are demonstrated.

Pyruvate Assay:

Pyruvate: The present invention provides for the measurement ofhomocysteine based on the use of the enzymes CBS and CBL to cyclehomocysteine with the production of pyruvate and ammonia. The rate ofpyruvate and ammonia production was predicted to be proportional to theoriginal homocysteine concentration in the sample. Measurement of thisrate using appropriate enzyme(s) presents an opportunity for ahomogeneous homocysteine method.

In one aspect of the present invention a highly sensitive method forpyruvate quantitation has been developed. In the method pyruvate oxidaseconverts pyruvate to acetyl phosphate, carbon dioxide and peroxide.Peroxidase subsequently converts the peroxide, TOOS, and4-aminoantipyrine to a chromogen, which exhibits an absorption maximumnear 550 nm. The molar absorptivity of this chromogen is quite high,about 36 L mol-1 cm-1.

Using reagent components shown in Table 1 and the Cobas FARA (Roche,Basel, Switzerland) instrument parameters listed in Table 2, pyruvatewas easily measured in the μM range by an early-read blank and endpointreaction in about 5-10 minutes. Absorbance versus time plots for aqueouscalibrators are shown in FIG. 1. An actual calibration curve is shown inFIG. 2.

TABLE 1 Pyruvate Method: Reagent Components Chemical ConcentrationReagent 1 HEPES, hemisodium salt 21.5 mM HEPES, Acid 28.6 mM EDTA (4Na)5.0 mM Mg SO₄.7H2O 49.8 mM K₂HPO₄, dibasic 7.5 mM TOOS 1.4 mM TTHA 0.8mM TPP 0.2 mM 4-aminoantipyrine 1.0 mM BSA 1.9 g/L Potassiumferrocyanide 0.07 mM Peroxidase, horseradish 3.0 KU/L Pyruvate Oxidase3.0 KU/L pH = 7.0

TABLE 2 Pyruvate Method: Cobas FARA parameters Parameter Setting GENERALmeasurement mode ABS reaction mode P-A Calibration mode SLOPE AVGreagent blank REAG/DIL Wavelength 550 nm Temperature 37° C. ANALYSISsample volume 40 μL diluent volume 10 μL diluent name H₂O Reagent volume250 μL  incubation time 60 s start reagent volume na diluent name H₂Odiluent volume na incubation time na start reagent 2 volume na diluentname H₂O diluent volume na Temperature delay na ABSORBANCE READINGSFirst 0.5 sec Number 14  Interval  30 sec CALCULATION Number of steps 1calculation step A KINETIC First 1 Last 14  CALIBRATION Number of stds 1STD1 50 μM Replicate 2Cystathionine Assay:

Cystathionine was measured by adding CBL to the pyruvate reagentdescribed above. CBL converts cystathionine to homocysteine, pyruvateand ammonia. The pyruvate was then measured as described above. Thecomposition of the reagent used to measure cystathionine is shown inTable 3. The measurement can be made using any suitable instrument. Inthe present example a Cobas FARA (Roche, Basel, Switzerland) was used.Typical reaction parameters are shown in Table 4. FIG. 3 depictsabsorbance vs. time plots obtained using aqueous solutions ofcystathionine. The reaction reaches endpoint in less than ten minutes.

TABLE 3 Cystathionine Method: Reagent Components Chemical ConcentrationReagent 1 HEPES, hemisodium salt 20.3 mM HEPES, Acid 27.0 mM EDTA (4Na)4.7 mM Mg SO4.7H2O 47.1 mM K2HPO4, dibasic 7.1 mM TOOS 1.3 mM TTHA 0.8mM TPP 0.2 mM 4-aminoantipyrine 0.9 mM BSA 1.8 g/L Potassiumferrocyanide 0.07 mM Peroxidase, horseradish 2.8 KU/L Pyruvate Oxidase2.8 KU/L CBL 1.9 g/L pH = 7.0

TABLE 4 Cystathione Method: Cobas FARA parameters Parameter SettingGENERAL measurement mode ABS reaction mode P-A calibration mode LININTER reagent blank REAG/DIL wavelength 550 nm temperature 37° C.ANALYSIS sample volume 30 μl diluent volume 10 μl diluent name H₂Oreagent volume 260 μl  incubation time 60 sec start reagent volume nadiluent name H₂O diluent volume na incubation time na start reagent 2volume na diluent name H₂O diluent volume na temperature delay naABSORBANCE READINGS first  5 sec number 21  interval 60 sec CALCULATIONnumber of steps 1 calculation step A ENDPOINT first 1 last 10 CALIBRATION number of stds 1 STDI 50 μM replicate 2

FIG. 4 depicts standard curves obtained using aqueous solutions ofcystathionine. These curves are linear over at least the cystathionineconcentration range of 0-100 μM. The chromophore formed was stable overapproximately 15-20 minutes. However, if samples containing 10-20 foldhigher cystathionine concentrations were assayed, some instability ofthe chromophore was evident.

In some experiments serine was included in the reaction mixture. Serineat a level of 250 μM exhibited no significant effect. Higher levels ofserine were also tolerated by this assay. The non-interference of serineis an important observation because the presence of serine is necessaryfor the assay of homocysteine.

EXAMPLE 6 Enzyme Cycling Assays for Homocysteine

In this example the enzyme cycling systems for determining theconcentration of homocysteine are provided using either pyruvateoxidase/peroxidase signaling system or lactate dehydrogenase signalingsystem.

Demonstration of Cycling: CBS/CBL/PO/HRP Enzyme Cycling System

The cycling system has been demonstrated wherein the reaction wasinitiated using either homocysteine or cystathionine. The reagentcomponents used in these experiments are shown in Table 5. CBS andserine were added separately following mixing of the sample with reagent1 which contains all other components. Pertinent Cobas FARA parametersare shown in Table 6. Cystathionine or homocysteine was dissolved inwater to prepare standards at appropriate concentrations. Time versusabsorbance plots are shown in FIGS. 5 and 6. After a lag time of threeto six minutes the plots are linear. A typical homocysteine calibrationcurve (FIG. 7) which, while not completely linear, can be used toquantify homocysteine concentrations in suitable specimens using anappropriate curve-fitting technique.

TABLE 5 CBS/CBL Cycling, PO/HPR Method Reagent Components ChemicalConcentration Reagent 1 HEPES, hemisodium salt 20.3 mM HEPES, Acid 27.0mM EDTA (4Na) 4.7 mM Mg SO₄.7H2O 47.1 mM K₂HPO₄, dibasic 7.1 mM TOOS 1.3mM TTHA 0.8 mM TPP 0.2 mM 4-aminoantipyrine 0.9 mM BSA 1.8 g/L Potassiumferrocyanide 0.07 mM Peroxidase, horseradish 3.0 KU/L Pyruvate Oxidase3.0 KU/L CBL pH = 7.0 Reagent SR1 Serine 3.1 mM Reagent SR2 CBS 1.23 g/L

TABLE 6 CBS/CBL/PO/HPR cycling system: Cobas FARA parameters ParameterSetting GENERAL measurement mode ABS reaction mode P-I-SR1-I-SR2-ACalibration mode LIN INTER reagent blank REAG/DIL Wavelength 550 nmTemperature 37° C. ANALYSIS sample volume 30 μl diluent volume 10 μldiluent name H₂O reagent volume 260 μl  Incubation time  60 sec startreagent volume 30 μl (serine) diluent name H₂O diluent volume  5 μlIncubation time  10 sec start reagent 2 volume 30 μl (CBS) diluent nameH₂O diluent volume  5 μl temperature delay na ABSORBANCE READINGS First0.5 sec Number 14  Interval  30 sec CALCULATION number of steps 1Calculation step A ENDPOINT or KINETIC First 1 Last 14  CALIBRATIONnumber of stds 1 STD1 50 μM Replicate 2Reduction of Disulfide Bonds:

Very little homocysteine is present in the reduced, nondisulfide bondedstate in human plasma. Most homocysteine is coupled to proteins or smallmolecules through disulfide bonds. Disulfide reduction of thesecompounds is necessary to liberate homocysteine for measurement by anymethod for total homocysteine determination. This example demonstratesthat use of a reducing agent is compatible with a system using lactatedehyrdrogenase and NADH. Potential candidate reducing agents includeDTE, DTT, n-acetylcysteine, thioglycolic acid, TCEP and the like. Foruse of these compounds in the method of the present invention, preciseadjustment of reducing agent concentration and instrument parameters arenecessary. Early indications that certain reducing agents could hinderthe action of pyruvate oxidase and or horseradish peroxidase led to theinvestigation of the alternative pyruvate detection system describedbelow.

Demonstration of Cycling: CBS/CBL/LDH System:

Pyruvate reacts with NADH in the presence of the enzyme lactatedehydrogenase to produce lactic acid and NAD. Pyruvate can be quantifiedby measuring the absorbance decrease at 340 nm. The molar absorptivityof NADH is about six-fold less than that of the peroxidase generatedchromogen. However, this does not effect the system of the presentinvention because CBL/CBS homocysteine cycling is able to generaterelatively large amounts of pyruvate.

The reagent system used to generate the data presented in FIGS. 8, 9,10, & 11 comprises serine, CBL and lactate dehydrogenase in HEPES buffer(pH 7.2) as the first reagent. NADH in TRIS buffer (pH 8.5) and CBS arethen added in sequence. All of the components can, however, be combineddifferently to form either a two or three reagent homocysteinemeasurement system. Reagent concentrations are shown in Table 7, andCobas FARA parameters are shown in Table 8. The NADH concentration wasadjusted to provide sufficient linearity while allowing for plasmasample absorption at 340 nm.

TABLE 7 CBS/CBL/LDH CYCLING SYSTEM: Reagent Components ChemicalConcentration Reagent 1 HEPES, hemisodium salt 39.4 mM HEPES, acid 12.6mM Serine 0.72 mM Lactate dehydrogenase 33,000 U/L CBL 6.05 ug/L pH =7.2 Reagent SR1 NADH 4.16 mM TRIS 50 mM pH = 8.5 Reagent SR2 CBS 996mg/L TRIS 50 mM Sodium Chloride 100 mM pyridoxal phosphate 5 uM SampleDiluent DTT 13 mM HEPES, hemisodium salt 56.7 mM HEPES, Acid 18.1 mM pH= 7.2

TABLE 8 CBS/CBL/LDH cycling system: Cobas FARA parameters ParameterSetting GENERAL measurement mode ABS reaction mode P-T-I-SR1-I-SR2-Acalibration mode LIN INTER reagent blank REAG/DIL Wavelength 340 nmtemperature 37° C. ANALYSIS sample volume 30 μl diluent volume 10 μldiluent name H₂O (or reducing agent) reagent volume 250 μl  incubationtime 60 sec start reagent volume 15 μl (NADH) diluent name H₂O diluentvolume  5 μl incubation time 10 sec start reagent 2 volume 30 μl (CBS)diluent name H₂O diluent volume 10 μl temperature delay NA ABSORBANCEREADINGS First  5 sec Number 16  Interval 30 sec CALCULATION Number ofsteps 1 calculation step A ENDPOINT or KINETIC First 6 Last 20 CALIBRATION Number of stds 1 STD1 25 or 50 μM Replicate 2Analysis of Aqueous Solutions:

The system performed well for the detection of homocysteine in aqueoussolution. Typical absorption versus time plots are shown in FIGS. 8 and9. These plots were linear with little or no apparent lag phase. Ratesof NADH disappearance were relatively high. Moreover, the calibrationplot of homocysteine concentration versus absorbance was linear over therange of 0-100 μM in the sample (FIG. 10). Thus, only one calibrator,e.g. 25 μM, together with a reagent blank was necessary for calibration.

Homocysteine solutions were prepared at various concentrations todemonstrate performance of the method over a homocysteine concentrationrange of 2-20 μM. Results are shown in Table 9 both in the presence andabsence of DTT. As mentioned above, DTT serves to reduce disulfide bondsin samples (eg serum, plasma, etc.). DTT does not significantlyinterfere with the lactate dehydrogenase catalyzed reaction.

TABLE 9 Demonstration of Linearity: CBS/CBL/LDH cycling system Reactiontime = 3 min. Observed value (uM) Homocysteine conc. (uM) (−) DTT (+)DTT 2 2 1.2 5 5.2 4.3 8 8.2 7.4 10 10.4 9.9 12.5 12.7 12.5 16.7 16.816.3 20 20.5 20Analysis of Human Plasma Based Control Products:

Accurate performance of the CBS/CBL/LDH cycling system has beendemonstrated herein using human plasma based homocysteine controlproducts manufactured by the Bio-Rad Co. Various concentrations of DTThave been used, and incubation times have been varied in order toachieve optimum homocysteine recovery. Incubation times of 5-15 min withDTT concentrations of 1.6-3.2 mM gave homocysteine measurements wellwithin the ranges specified for various other methods.

Human Plasma Samples:

The homocysteine cycling assay has demonstrated good performance usinghuman plasma samples with DTT as the reducing agent. Results agree wellwith those obtained by an independent laboratory using the Abbott IMxhomocysteine method (FIG. 11). Cobas FARA parameters used in thisexperiment are shown in Table 10 and reagent components are shown inTable 7. In this assay an aqueous homocysteine calibrator (25 uM) wasused. Absorbance vs. time plots for aqueous based samples were linear inthe absence of DTT and nearly linear in the presence of DTT. However,plots obtained using plasma samples were different, i.e., not linearafter the first three minutes. Under the conditions outlined in Table 7and Table 10, cycling appears to gradually slow down over time.Nevertheless, the correlation study presented in FIG. 11 demonstratesrelatively close agreement between the present method (outlined in Table7 and Table 10) with those obtained by IMx when testing human serumsamples.

TABLE 10 CBS/CBL/LDH cycling system: Cobas FARA parameters for humanplasma correlation study Parameter Setting GENERAL measurement mode ABSreaction mode P-T-I-SR1-I-SR2-A Calibration mode LIN INTER reagent blankREAG/DIL Wavelength 340 nm Temperature 37° C. ANALYSIS sample volume 30μl diluent volume 10 μl diluent name H₂O (or reducing agent) Temperaturedelay 900 sec  reagent volume 250 μl  Incubation time 60 sec startreagent volume 15 μl (NADH) diluent name H₂O diluent volume  5 μlIncubation time 10 sec start reagent 2 volume 30 μl (CBS) diluent nameH₂O diluent volume 10 μl Temperature delay NA ABSORBANCE READINGS First 5 sec Number 24  Interval 30 sec CALCULATION number of steps 1Calculation step A ENDPOINT or KINETIC First 1 Last 6 CALIBRATION numberof stds 1 STD1 25 or 50 μM Replicate 2

In a preferred embodiment, the rates were measured in the first threeminutes after reaction initiation. Additional optimization of thesystem, including variation in salt and detergent concentrations, willlikely result in more consistent performance with aqueous and plasmasamples. The assay has been equally accurate when components werecombined into two or three mixtures of reagents.

EXAMPLE 7 Comparison of Homocysteine Assays

In this example, a preferred homocysteine assay in accordance with theinvention was tested and compared with two existing assays, namely aconventional HPLC technique and the commercially available IMx assay.

The assay of the invention was carried out using three reagents as shownin Table 11. The first reagent R1 was initially in the form of powderand was reconstituted to a level of 1.5 g powder per 100 mL water. Theoverall assay kit also included calibrators containing L-cystathioninespiked at various levels in a human plasma matrix, as well as twocontrol samples made up in a processed human plasma matrix.

TABLE 11 CBS/CBL/LDH CYCLING SYSTEM: Reagent Components ChemicalConcentration Reagent 1 HEPES, hemisodium salt 43.3 mM Hepes, acid 12.7mM Serine 1.295 mM Lactate dehydrogenase >800 U/L NADH 1.06 mM TritonX-100 0.05% v/v Sodium azide 7.7 mM pH = 8.0 Reagent SR1 DTE 6.75 mMCitric acid 20 mM pH = 2.0 Reagent SR2 CBS 18.7 KU/L CBL 8.9 KU/LSorbitol 1.65 M Sodium chloride 100 mM Pyrdoxal phosphate 5 uM Sodiumazide 7.7 mM TRIS/HCl 50 mM pH = 8.0

TABLE 12 CBS/CBL/LDH Cycling System: Cobas MIRA Parameters ParameterSetting GENERAL Measurement Mode Absorb Reaction Mode R-S-SR1-SR2Calibration Mode Lin Regre Reagent Blank Reag/Dil Cleaner No Wavelength340 nm Decimal Position 2 Unit umol/L ANALYSIS Post Dil. Factor No Conc.Factor No Sample cycle 1 volume 30.0 uL Diluent name H2O volume 25.0 uLReagent cycle 1 volume 90 uL Start R1 cycle 2 volume 25 ul Diluent nameH2O volume 25 uL Start R2 cycle 17 volume 35 uL Diuent name H2O volume15 uL CALCULATION Sample Limit No Reac. Direction Decrease Check OffAntigen Excess No Covers. Factor 1.00 Offset 0.00 Test range Low On HighOn Normal range Low No High No Number of Steps 1 Calc. Step A KineticReadings First 18 Last 32 Reaction Limit No CALIBRATION Calib. IntervalEach Run Blank Reag. Range Low No High No Blank Range Low No High NoStandard Position 1 1 3.5 umol/L 2 7.0 umol/L 3 17 umol/L 4 27 umol/L 547 umol/L 6 No 7 No Replicate Single Deviation Correction Std. NoControl CS1 position No CS2 position No CS3 position No

In performing this assay, the commercially available Cobas MIRAinstrument was used. The Cobas MIRA software allows for 50 programable,25 second cycles. In particular, the instrument was programmed accordingto the parameters in Table 12. In cycle 1, 30 μL of the sample was addedto a cuvette, followed by 25 μL of flush water and 90 μL of liquidreagent R1. At the beginning of cycle 2, 25 μL of reagent SR1 was addedwith an additional 25 μL of flush water. Thereupon, the mixture wasallowed to incubate for 15 cycles (6.25 min.) to allow the reductant toliberate any bound homocysteine and to destroy any endogenous pyruvatepresent. At this point, 35 μL of the CBS/CBL enzyme reagent was addedwith an additional 15 μL of flush water. The decrease in absorbance at340 nm was measured over time between cycles 18 and 32 as a measure ofhomocysteine in the sample. The total assay time between sample additionand the final absorbance reading was 32 cycles, or 13 min. and 20 sec.Since multiple samples are run in parallel, additional sample resultsare produced by the MIRA at a rate of approximately one every 30-90seconds depending on the length of the run and the number of instrumentcalibrators used. Using similar programming parameters, this assay maybe adapted to even faster instruments (eg. Roche INTEGRA, Hitachi, ACE,etc) which could yield even greater throughput rates.

A total of 24 samples were tested using the assay of the invention andthe comparative known assays. Table 13 below sets forth the homocysteineconcentrations obtained using these three methods. FIGS. 12-14 arerespective correlation graphs depicting graphically the results of Table13. As can be seen, the method of the invention correlates closely withboth IMx and HPLC; with closer agreement typically seen with IMx.However, the present method is substantially less expensive, can be runfaster, and can be performed using generally available chemicalanalyzers.

The invention exhibits very good between-run precision as shown in Table14. Typical within-run CV's were found to be less than 4% overhomocysteine concentrations of about 7-20 μmol/L.

TABLE 13 Comparison of Homocysteine Assays Homocysteine Concentration[μM] Sample # HPLC Invention IMx 1 9.5 11.5 11.7 2 14.3 13.1 12.1 3 8.87.8 7.5 4 10.6 10.1 9.4 5 10.5 10.1 9.9 6 8.6 9.2 7.2 7 7.6 5.8 6.2 85.2 3.5 3.1 9 9.6 9.3 9.7 10 7.7 8.7 7.5 11 11.2 11.1 9.4 12 10.5 8.38.1 13 11.9 8.4 8.3 14 10.8 9.2 8.9 15 14.1 12.3 12.5 16 24.3 16.3 17.917 20.9 17.7 17.8 18 24.2 22.7 22.1 19 33.6 26.4 26.4 20 21.0 20.7 18.921 22.7 19.6 18.1 22 20.5 17.3 16.6 23 25.6 23.3 22.0 24 20.5 17.0 15.8Average 15.2 13.3 12.8 Intercept [HPLC] 1.4 0.9 Slope [HPLC] 1.2 1.2 R[HPLC] 0.957 0.974 Intercept [IMx] -0.3 0.5 Slope [IMx] 0.8 1.0 r [IMx]0.976 0.990

TABLE 14 Between-Run Precision Homocysteine (μmol/L) Sample Run 1 Run 2Average % CV 1 16.35 16.46 16.41 0.47 2 12.13 12.58 12.36 2.58 3 19.4521.6 20.53 7.41 4 16.29 16.79 16.54 2.14 5 11.13 10.01 10.57 7.49 6 7.337.56 7.45 2.18 7 6.31 7.24 6.78 9.71 8 7.13 5.54 6.34 17.75 9 14.87 14.514.69 1.78 10 13.81 13.41 13.61 2.08 11 26.86 28.66 27.76 4.58 12 12.4513.05 12.75 3.33 13 10.15 10.74 10.45 3.99 14 9.6 10.39 10.00 5.59 1512.53 13.67 13.10 6.15 16 14.60 15.20 14.90 2.85 17 25.03 25.65 25.341.73 18 20.77 20.03 20.40 2.56 19 31.83 32.10 31.97 0.60 20 36.86 36.4336.65 0.83 21 11.65 11.48 11.57 1.04 22 40.22 38.25 39.24 3.55 23 31.6832.62 32.15 2.07 24 7.68 6.38 7.03 13.08 Average CV (%) = 4.22

EXAMPLE 8 Effect of Reagent Concentration Variations

In this example, the preferred assay of the invention described inExample 7 was performed with variations in some of the reagentcomponents, namely LDH, NADH and serine. This was done in order todetermine the optimum amounts for these reagent components. The assayswere performed as described above, with the respective components variedin concentration. Table 15 sets forth the results from these tests.

TABLE 15 Effect of Reagent Component Concentration Variations Target[LDH] (U/L) Homocysteine Final range of Reaction SLOPE INTERCEPT BLANKcontrol sample: Mixture (mA/min/uM) mA/min mA/min 25-35 μM 114.5 1.21−0.14 −10.2 25.8 229.3 1.14 0.12 −11.2 28.1 458.7 1.17 0.14 −11.3 26.6917.5 0.88 1.89 −11.3 27.2 [NADH] (mM) 0.130 0.77 4.23 −10.7 32.9 0.2591.12 0.05 −11 25.2 0.389 1.09 0.45 −10.7 26.8 0.519 1.13 0.49 −11.1 26.0[SERINE] (mM) 0.125 0.64 2.23 −6.7 30.9 0.25 0.92 1.26 −9.0 28.1 0.51.13 0.63 −10.8 27.1 1 1.16 0.15 −13.6 28.3 2 1.23 0.33 −15.4 28.2

The results shown in Table 15 enabled the selection of final reagentcomponent levels which provided adequate sensitivity with a minimalblank rate while also trying to minimize reagent costs.

EXAMPLE 9 Effect of R1 Formulation Change on the Cycling Assay

This example determined the effect of altering the pH by changing thebuffer to TRIS, 250 mM at a pH of 8.4 and by adding lipase andα-cyclodextrin to Reagent 1. The performance of the assay was thentested after making these formulation changes. Table 16 details thereagent components used for these experiments.

TABLE 16 CBS/CBL/LDH CYCLING SYSTEM: Reagent Components ChemicalConcentration Reagent 1 TRIS 250 mM α-cyclodextrin 0.1% Lipase ≧200 kU/LSerine 1.295 mM Lactate dehydrogenase >800 U/L NADH 1.06 mM Triton X-1000.05% v/v Sodium azide 7.7 mM pH = 8.2 Reagent SR1 TCEP 26.3 mM ReagentSR2 CBS 28.5 KU/L CBL 9.4 KU/L Glycerol 10% Sorbitol 1% Pyridoxalphosphate 5 μM Sodium azide 7.7 mM Potassium phosphate 100 mM pH = 7.6

By changing the buffer of the Reagent 1 to TRIS, 250 mM at pH 8.4,improved assay consistency. This was because CBS and CBL aresubstantially more active at pH 8.4 and the rate of cycling is increasedby about a factor of three.

Lipase and α-cyclodextrin were added to combat the problems associatedwith turbid (lipemic) samples which are sometimes encountered in theclinical laboratory. Such samples may arise when the patient from whomthe sample is drawn has eaten shortly before the blood sample is drawnor when the patient has certain disease processes going on. Turbiditycan interfere with results of this testing by producing such a highabsorbance at 340 nm that the instrument cannot accurately measure thesmall changes in absorbance produced by the conversion of NADH to NAD.Turbidity can also interfere with the results of this testingdynamically by changing over time during the time when the NADH to NADconversion is measured. For example, if the amount of turbidity isdecreasing, the absorbance at 340 nm also decreases because lightscattering is less. This effect adds to the absorbance decrease causedby the reduction of pyruvate and the resulting homocysteinedetermination may be substantially too high. The addition of lipase andα-cyclodextrin to R1 did decrease the turbidity of samples, presumablyby the hydrolysis of triglycerides to glycerol and free fatty acids bylipase and by the free fatty acids forming complexes with theα-cyclodextrin. Thereby, turbid reaction mixtures clear by the time thatSR2 (R3) is added allowing for accurate measurement of the rate ofpyruvate production. Absorbances at 340 nm of many turbid reactionmixtures did rapidly decrease and level off in several minutes.Homocysteine results were accurate. However, the clearing of some turbidsamples was not complete before the addition of SR2 (R3) and therefore,the results for some samples were somewhat inaccurate. Nonetheless, theuse of lipase and alpha-cyclodextrin appears to be a very promisingapproach for achieving accurate results for turbid samples.

In addition to the changes made in the reagent constituents, the volumeswere also varied in order to assess their effects on the performance ofthe assay. These changes are reflected in Table 17, which shows atypical cycling parameter on the Cobas MIRA. Following these changes acorrelation study was carried out on 86 human samples with the resultsshown in Table 18.

TABLE 17 CBS/CBL/LDH Cycling System: Cobas MIRA Parameters ParameterSetting GENERAL Measurement Mode Absorb Reaction Mode R-S-SR1-SR2Calibration Mode Linear Regression Reagent Blank Reag/Sol Cleaner NoWavelength 340 nm Decimal Position 2 Unit μmol/L ANALYSIS Post Dil.Factor No Conc. Factor No Sample cycle 1 volume 20.0 μL Diluent name H₂Ovolume 45.0 μL Reagent cycle 1 volume 90 μL Start R1 cycle 2 volume 21μL Diluent name H₂O volume 26 μL Start R2 cycle 17 volume 13 μL Diluentname H₂O volume 10 μL CALCULATION Sample Limit No Reac. DirectionDecrease Check Off Antigen Excess No Covers. Factor 1.00 Offset 0.00Test range Low On High On Normal range Low No High No Number of Steps 1Calc. Step A Kinetic Readings First 20 Last 31 Reaction Limit NoCALIBRATION Calib. Interval Each Run Blank Reag. Range Low No High NoBlank Range Low No High No Standard Position 1 1 0.0 μmol/L 2 8.5 μmol/L3 17.2 μmol/L 4 25.2 μmol/L 5 43.0 μmol/L 6 No 7 No Replicate DuplicateDeviation Correction Std. No Control CS1 position No CS2 position No CS3position No

TABLE 18 Correlation Data with Improvement listed in Tables 16 and 17.IMx Catch Sample (μmol/L) (μmol/L) 2-01 11.0 12.0 2-02 17.7 17.3 2-0318.6 18.4 2-04 22.1 21.2 2-05 12.2 13.8 2-06 15.2 14.9 2-07 17.6 20.42-09 51.8 49.9 2-11 29.1 28.2 2-13 19.6 19.2 2-15 25.0 24.7 2-16 22.923.7 2-17 21.9 21.3 2-18 25.0 23.80 2-19 22.2 22.10 2-21 23.6 20.80 2-2213.0 13.60 2-23 23.1 21.80 2-24 17.0 17.60 2-26 15.9 14.80 2-27 8.8 9.302-28 9.1 9.40 2-29 5.2 5.40 2-30 8.2 8.10 2-31 4.7 5.40 2-32 6.6 6.802-33 7.8 8.10 2-34 7.0 7.0 2-35 6.9 7.3 2-36 4.8 5.7 4-01 7.2 7.3 4-027.3 9.1 4-03 6.2 5.8 4-04 9.5 9.5 4-05 6.2 7.4 4-06 8.2 8.0 4-07 6.8 6.74-08 8.1 7.4 4-09 6.5 7.9 4-10 7.5 8.5 4-11 8.1 8.6 4-12 6.1 5.7 4-136.19 6.20 4-14 5.31 5.90 4-15 7.17 6.80 4-16 5.05 7.00 4-17 5.93 7.804-18 8.57 8.10 4-19 4.87 5.70 4-21 19.37 19.50 4-22 8.61 9.30 4-23 11.0212.50 4-24 12.75 13.30 4-25 12.28 12.00 4-26 14.63 14.30 4-27 9.33 11.304-29 18.39 19.60 4-30 14.13 14.20 4-31 8.63 8.60 4-32 9.95 9.80 4-338.08 10.20 4-34 12.34 12.00 4-35 12.16 13.10 4-36 15.75 16.30 4-37 14.3714.30 4-38 14.72 15.20 4-39 10.26 11.20 4-40 12.26 14.00 4-41 24.6225.00 4-43 27.25 29.50 4-46 21.26 19.60 4-47 22.08 23.30 4-50 15.1415.50 El 4.74 4.90 E2 8.24 8.00 E3 5.10 5.30 E6 13.79 14.50 E8 8.7910.00 E9 10.57 11.50 Ell 7.10 7.40 E13 7.65 7.30 E14 12.19 12.30 E156.47 6.14 E16 5.28 4.70 E20 7.93 7.80 Av. 12.6 12.9 Slope 0.957Intercept 0.8135 R 0.9927

As shown by this comparison table, the results of this assay with thechanged formulation was very accurate when compared with thecommercially available IMx assay.

EXAMPLE 10 Use of Other Detergents and Their Concentration Variation

In this example, different detergents were used in combination with EDTAas components of R1. EDTA is known to stabilize lipid particles. Againthe goal was to achieve accurate results when samples are turbid. Inparticular, the detergents Brij-35 and Genapol X-80 were studied. It wasfound that EDTA was particularly effective at the concentration of 2.5mM to help minimize non-specific decrease in absorbance due to turbiditychanges when lipemic samples are analyzed. Detergent concentrations werealso varied as shown in Tables 19 and 20. One preferred composition forreagent R1 is shown in Table 21. Brij 35 was present at the level of0.025% v/v and EDTA was present at 2.5 mM. Lipase and alpha-cyclodextrinwere not present because they do not seem to be necessary to achieveaccuracy with turbid samples when EDTA is present. Brij-35 (0.025%) wasalso put into SR2 (R3) in the place of Triton X-100. This may be apromising substitution because Triton X-100 is considered to be anenvironmental hazard by some countries. Table 22 shows the results ofcorrelation study performed with the preferred reagent components.

TABLE 19 Varying Genapol X-80 Concentration Results (μmol/L) change ofGenapol X-80 I M x concentration in R1 Samples (μmol/L) 0.1% 0.3% 0.5%Low 7.00 6.31 6.19 5.64 Medium 12.50 11.96 11.48 10.37 High 25.00 23.0822.96 22.86 D910 14.30 14.38 14.50 14.11 D912 9.40 9.41 10.60 9.50 D9135.00 5.58 6.47 4.53 D915 21.20 23.85 21.93 20.92 PBI 27 7.40 7.69 7.706.82 PBI 28 6.80 7.33 7.19 5.96 PBI 29 7.10 7.83 7.50 6.85 PBI 10 8.909.61 9.07 7.67 MAS 1 5.90 4.63 4.45 4.15 MAS 2 14.50 14.46 15.33 14.47MAS 3 23.10 22.60 23.07 23.65 D913X2 2.50 2.45 2.46 2.17 Average 11.3711.41 11.39 10.64

TABLE 20 Varying Brij-35 Concentration in R1 Catch Results (μmol/L) as afunction IMx of Brij-35 Concentration in R1 Samples (μmol/L) 0.025%0.05% 0.1% 0.5% 1 5.88 5.03 5.35 5.26 5.09 2 14.57 15.46 16.20 15.9215.60 3 23.52 24.66 24.89 23.67 24.02 2 6.49 6.80 7.04 8.44 10.74 1810.15 10.72 11.29 9.95 10.42 21 4.62 4.86 5.84 5.73 4.96 32 8.90 8.178.38 8.67 8.43 Average 10.59 10.81 11.28 11.09 11.32

TABLE 21 CBS/CBL/LDH CYCLING SYSTEM: Reagent Components ChemicalConcentration Reagent 1 TRIS 250 mM EDTA 2.5 mM Serine 1.295 mM Lactatedehydrogenase >800 U/L NADH 1.06 mM Brij 35 0.025% w/v Sodium azide 7.7mM pH = 8.2 Reagent SR1 TCEP 26.3 mM Reagent SR2 CBS 28.5 KU/L CBL 9.4KU/L Glycerol 10% Sorbitol 1% Pyridoxal phosphate 5 μM Sodium azide 7.7mM Potassium phosphate 100 mM pH = 7.6

TABLE 22 Correlation Data with best improvement IMx Catch Sample(μmol/L)L) (μmol/ 1 5.90 5.15 2 14.57 15.36 3 23.52 23.52 701 17.9517.03 702 11.51 11.55 703 11.81 12.97 704 10.13 10.64 705 22.37 21.87706 16.97 16.79 707 7.76 7.76 708 9.35 9.90 709 7.33 6.60 710 7.24 6.90711 8.50 8.54 712 19.93 19.00 713 6.82 6.04 715 6.91 6.93 716 7.35 7.05717 9.44 9.26 718 14.16 15.61 719 5.73 5.25 720 6.32 6.00 721 11.3012.75 722 16.23 17.55 723 11.77 12.11 724 8.39 9.72 725 10.17 11.48 7266.69 6.14 727 8.01 7.50 728 10.45 10.64 729 11.73 11.27 730 14.39 14.87731 8.61 8.76 732 7.56 7.67 733 7.35 7.56 734 6.54 8.34 735 8.39 7.91Av. 10.79 10.92 slope 1.0035 intercept 0.0195 R 0.9873

When Brij-35 and EDTA were substituted for Triton X-100 and lipase plusα-cyclodextrin, there was some clearing of the turbid reaction mixtures.However, perhaps not as much as in the case when lipase andalpha-cyclodextrin are also used. Importantly, the absorbances at 340 nmquickly stabilized with zero rates of change. Thus when SR2 (R3) wasadded, the rate of pyruvate production is accurately measured andaccurate results are obtained even in the cases of samples appearinglike milk. This is even the case when the time between addition of R1and SR2 (R3) is shortened by 2-4 minutes, which may be necessary toapply the homocysteine assay of the present invention to other automatedinstruments.

EXAMPLE 11 Results from Changing Reducing Agent to TCEP and Alterationsin Concentration of TCEP

For this example, the reducing agent DTE was replaced with Tris(2-carboxyethylphosphine) hydrochloride (TCEP). TCEP has the advantageof being stable in purified water for an extended period of time.Replacement of DTE with TCEP reduced the reagent blank reaction fromabout 12 to 15 mA/min to about 4 to 5 mA/min. The lower reagent blankenables better Homocysteine assay precision. To find the optimumconcentration of TCEP for this Homocysteine assay, a range ofconcentration was tested. The results from these experiments are givenin Table 23.

TABLE 23 Varying TCEP Concentration in SR1 Homocysteine Results (μmol/L)SR1 Catch Assay Formulation IMx A B C D E F MAS 1 5.88 6.37 6.18 5.485.51 4.42 4.17 MAS 2 14.57 16.49 15.67 15.95 16.03 15.80 14.77 MAS 323.52 23.19 21.98 23.33 25.11 24.02 23.09 PBI 20 7.2 5.81 6.24 7.81 8.006.95 8.33 PBI 22 7.8 7.61 7.66 8.28 8.69 8.44 8.23 PBI 36 5.1 4.16 4.614.71 5.05 5.18 4.71 PBI 39 4.6 3.11 4.25 4.38 4.47 3.68 4.09 Doreen 7097.33 6.68 6.64 6.71 7.28 6.23 6.43 Average 9.50 9.17 9.15 9.58 10.029.34 9.22 TCEP Concentrations: A = 6.3 mM; B = 13.85 mM; C = 26.44 mM; D= 32.74 mM; E = 39.03 mM; F = 52.88 mM

EXAMPLE 12 Determination of the Effect of Changing the R3 Buffer toPhosphate and the Addition of Glycerol to the Buffer

In order to have enzyme mixtures that would have longer stability andassay compatibility, the enzyme formulation was changed to a mixturethat provides significantly longer shelf-life than the previousformulation. The significant changes involved replacement of Tris bufferwith a phosphate and addition of glycerol to the buffer. Potassiumphosphate at concentrations ranging from 50 mM to 500 mM at pH 7.6 wereused and glycerol concentrations ranging from 0.5% to 15.0% (v/v) wereused. As a result from these changes, the shelf life was increasedsignificantly from weeks to more than a year at 4° C. In preferredforms, potassium phosphate can range from about 50 mM to about 250 mM.More preferably, potassium phosphate can range from about 75 mM to about150 mM. A particularly effective concentration of potassium phosphate inthis experiment was 100 mM. Glycerol can range from about 1-15%. Morepreferably, this range is from about 5-13%. A particularly effectiveconcentration of glycerol in this experiment was 10%.

EXAMPLE 13 Results Obtained Using Different Instruments

To determine the overall improvement resulting from all the changes tothe assay, precision evaluations were done on several instruments bywriting protocol that is specific to that instrument. Table 24 setsforth the parameters and settings used in accordance with the presentinvention with the Roche Hitachi 911 and Table 25 sets forth the sameparameters and settings when the Beckman CX-5 is used. Below are thetables showing the results of these precision tests. These results aresignificantly better than what was obtained with the earlierformulations of the assay. On the Hitachi 911 (Table 26) and Beckman CX5(Table 27) instruments, each sample shown was run 20× and the within runprecision was determined. Percent C. V. for samples as low as 5.92μmol/L was 3.31% and samples in the range of 23.01 μmol/L to 25.4 μmol/Lwas 1.9% to 2.03%. A total precision (Table 28) of between 7.3% and 2.6%was obtained on samples with homocysteine values of 4.6 μmol/L and 27.0μmol/L on the Cobas MIRA Plus. Each sample was run in duplicate, 2× perday for a total of 20 days.

TABLE 24 Catch Homocysteine Method Parameters for the Roche Hitachi 911Test THCY Assay Code Rate-A, 15 Wavelength (2nd/Primary) 376/340 AssayPoint 35-49-0-0 Serum S. Vol. (Normal) 28 S. Vol. (Decrease) 20 S. Vol.(Increase) 30 Urine S. Vol. (Normal) 10 S. Vol. (Decrease) 10 S. Vol.(Increase) 10 ABS. Limit 0-0-Decrease Prozone Limit 0-0-Lower Reagent T1200 μL - 0-0-00322-0 T2 25 μL - 0-0-00322-0 T3 0 - 0-0-00322-0Calibration Type Linear-2-2-0 Auto Time Out Blank 0 Span 0 2 Point 0Full 0 Auto Change Lot Cancel Bottle Cancel Select Tests via Keyboard:ENTER SD Limit 100 Duplicate Limit 1000 Sensitivity Limit 0 S1 ABS Limit(−32000) (32000) Compensated Limit Blank PARAMETER PAGE 2 Test THCY -00322 Test Name THCY Unit: μmol/L Data Mode On Board Report NameHomocysteine Control Interval 0 Instrument Factor (Y = aX + b) a 1 b 0Expected Values user defined Technical Limit Serum 0-10000 Urine(−99999) (999999) STD Conc.-Pos.-Sample-Pre.-Dil.-Calib. 10.0-18-28-0-0-501 2 25.2-26-28-0-0-026 3 0 4 0 5 0 6 0

For this experiment, T1 is prepared by mixing R1 and deionized water inthe ratio of 9 to 8.1. Reagents T2 and T4 correspond to Reagents SR1 andSR2, respectively.

TABLE 25 Catch Homocysteine Method Parameters for the Beckman CX-5Chemistry Name: HOMOCYSTEINE Test Name HOMO Calculation Factor: 0Reaction Type: RATE 1 Math Model: Linear Cal Time Limit: 24 Hrs ReactionDirection: Decreasing No. of Calibrators: 2 Units: μmol/L DecimalPrecision X.XX Primary Wavelength: 340 nm Secondary Wavelength: 380 nmSample Volume 25 μL Primary Inject Rgt: A 179 μL B 22 μL SecondaryInject Rgt: C 14 μL Add Time: 592 sec CALIBRATORS #1 0.00 #2 24.8MULTIPOINT SPAN 1-2: −0.001 REAGENT BLANK Start Read: 100 sec End Read:200 sec Low ABS Limit: −1.5 High ABS Limit 1.5 USABLE RANGE Lower Limit:0.00 Upper Limit: 99999.00 SUBSTRATE DEPLETION Initial Rate: −99.999Delta ABS 1.5For this example, Reagent A is prepared by mixing R1 and deionized waterin the ration of 9 to 8.1. Reagents B and C correspond to MIRA reagentsSR1 and SR2.

TABLE 26 Within Run Precision on Hitachi 911 Within Run Low Std. Dev.0.224 Mean = 8.3 μmol/L % C.V. 2.17 Medium Std. Dev. 0.324 Mean = 11.9μmol/L % C. V. 2.71 High Std. Dev. 0.516 Mean = 25.4 μmol/L % C. V. 2.03

TABLE 27 Within Run Precision on Beckman CX5 Within Run Low Std. Dev.0.196 Mean = 5.92 μmol/L % C.V. 3.31 Medium Std. Dev. 0.195 Mean = 11.12μmol/L % C. V. 1.70 High Std. Dev. 0.437 Mean = 23.01 μmol/L % C. V.1.90

TABLE 28 Total Precision on Roche MIRA-Plus Within Between Between RunRun Day Total Low Std. 0.31 0.14 0.04 0.34 Mean = 4.6 μmol/L Dev. % C.V.6.6 3.1 0.8 7.3 Medium Std. 0.40 0.08 0.23 0.47 Mean = 11.6 μmol/L Dev.% C.V. 3.5 0.7 2.0 4.0 High Std. 0.44 0.39 0.39 0.71 Mean = 27.0 μmol/LDev. % C.V. 1.6 1.5 1.4 2.6

EXAMPLE 14 Adaptability of the Assay to a 2-Reagent System

The present invention can also be adapted to system which uses only tworeagents. This makes the invention adaptable to wider range ofinstruments including those that can only use two reagents. Table 29shows the reagent contents of the two-reagent system and Table 30 showsthe MIRA plus parameters of how the assay was performed on thatchemistry analyzer. Shown in Table 31 are the comparison data obtainedusing the 3-reagents system versus the 2-reagents system.

TABLE 29 CBS/CBL/LDH CYCLING SYSTEM: Reagent Components for 2-ReagentsChemical Concentration Reagent 1 TRIS 250 mM EDTA 2.5 mM Serine 1.295 mMLactate dehydrogenase >800 U/L NADH 1.06 mM Brij 35 0.025% v/v Sodiumazide 7.7 mM TCEP 4.05 mM pH = 8.2 Reagent SR1 CBS 28.5 KU/L CBL 9.4KU/L Glycerol 10% Sorbitol 1% Pyrdoxal phosphate 5 μM Sodium azide 7.7mM Potassium phosphate 100 mM pH = 7.6

TABLE 30 CBS/CBL/LDH Cycling System for 2-Reagents: Cobas MIRAParameters Parameter Setting GENERAL Measurement Mode Absorb ReactionMode R-S-SR1 Calibration Mode Linear Regression Reagent Blank Reag/SolCleaner No Wavelength 340 nm Decimal Position 2 Unit μmol/L ANALYSISPost Dil. Factor No Conc. Factor No Sample cycle 1 volume 20.0 μLDiluent name H₂O volume 45.0 μL Reagent cycle 1 volume 137 μL Start R1cycle 16 volume 13 μL Diuent name H₂O volume 10 μL CALCULATION SampleLimit No Reac. Direction Decrease Check Off Antigen Excess No Covers.Factor 1.00 Offset 0.00 Test range Low On High On Normal range Low NoHigh No Number of Steps 1 Calc. Step A Kinetic Readings First 19 Last 30Reaction Limit No CALIBRATION Calib. Interval Each Run Blank Sol-Pos 1Reag. Range Low No High No Blank Range Low No High No Standard Position1 1 0.0 μmol/L 2 8.5 μmol/L 3 17.2 μmol/L 4 25.2 μmol/L 5 43.0 μmol/L 6No 7 No Replicate Duplicate Deviation Correction Std. No Control CS1position No CS2 position No CS3 position No

TABLE 31 Comparison of 2-Reagent System versus 3-Reagent System. CatchInc. Hcy Assay (μmol/L) IMx 2-Reagent 3-Reagent Samples (μmol/L) SystemSystem MAS 1 5.88 5.55 5.17 MAS 2 14.57 16.14 15.70 MAS 3 23.52 23.9923.47 P 10 8.90 9.32 9.65 P 11 7.60 7.45 7.27 P 33 7.80 8.11 8.29 P 345.93 5.21 5.69 754 + 848 16.00 15.68 16.15 Average 11.28 11.43 11.42

EXAMPLE 15 Adaptability of the Present Invention for Use withSingle-Point Calibration

The homocysteine assay of the present invention has also been adapted tousing a single calibration point, with the zero calibrator serving asthe blank. Table 32 below shows the parameters for performing a singlepoint calibration on the Mira instrument. The data below (Table 33)shows that using single point calibration in the slope average modegenerates results that are substantially equivalent to using themulti-point calibration curve.

TABLE 32 CBS/CBL/LDH Cycling System: Cobas MIRA Parameters for SinglePoint Calibration Parameter Setting GENERAL Measurement Mode AbsorbReaction Mode R-S-SR1 Calibration Mode Slope Average Reagent BlankReag/Sol Cleaner No Wavelength 340 nm Decimal Position 2 Unit μmol/LANALYSIS Post Dil. Factor No Conc. Factor No Sample cycle 1 volume 20.0μL Diluent name H₂O volume 45.0 μL Reagent cycle 1 volume 137 μL StartR1 cycle 16 volume 13 μL Diuent name H₂O volume 10 μL CALCULATION SampleLimit No Reac. Direction Decrease Check Off Antigen Excess No Covers.Factor 1.00 Offset 0.00 Test range Low On High On Normal range Low NoHigh No Number of Steps 1 Calc. Step A Kinetic Readings First 19 Last 30Reaction Limit No CALIBRATION Calib. Interval Each Run Blank Sol-Pos 1Reag. Range Low No High No Blank Range Low No High No Standard Position4 1 25.2 μmol/L 2 NO 3 NO Replicate Duplicate Deviation Correction Std.No Control CS1 position No CS2 position No CS3 position No

TABLE 33 Single-Point versus Multi-Point Calibration Catch Inc. HcyAssay (μmol/L) IMx Single Point Multi-Point Samples (μmol/L) CalibrationCalibration MAS 1 5.88 5.03 5.17 MAS 2 14.57 15.18 15.70 MAS 3 23.5222.97 23.47 PB1 10 8.90 8.82 9.65 PBI 11 7.60 8.26 7.27 PBI 33 7.80 8.058.29 PBI 34 5.93 5.80 5.69 754 + 848 16.00 15.17 16.15 Average 11.2811.16 11.42

EXAMPLE 16 Study of Homocysteine Assays Described in WO 00/28071

PCT Publication WO 00/28071 describes in detail two homocysteine assays.In the “first embodiment”, the assay is performed without a reductionstep. To convert that method to one useful for human plasma homocysteinemeasurements, either a separate off-line or a homogeneous reduction stepmust be incorporated. Publication WO 00/28071 describes a separateoff-line reduction step perhaps because a pyruvate oxidase cyclingsystem is used. That step entails an incubation of 20 min. before thereduced specimen can be analyzed, which greatly increases total assaytime and labor cost. Moreover, if the sample is to be assayed for morethan one analyte in the clinical laboratory, the primary serum or plasmasample must be split into two aliquots, one for the homocysteine testand one for any other ordered laboratory test. In contrast, the presentinvention enables homogeneous reduction since a LDH cycling system isused (Example 7).

Here, in Example 16 (Table 34), it is shown that DTT in fact doesinterfere with the pyruvate oxidase detection system described in the“second embodiment” of WO 00/28071. In particular, the reagent and DTTcompositions described in the “second embodiment” were used, and assayswith and without DTT were carried out against known pyruvate standards(12.5, 25, and 50 μmol/L) using a Cobas FARA instrument. In the case ofthe DTT test, 40 μL of each standard was mixed with 30 μL of the DTTsolution, followed by incubation for 100 sec. Thereafter, 90 μL ofpyruvate oxidase reagent was added and incubated for 30 sec. Absorbancereadings were then taken at 30, 60, 90, 120, 150, 180 and 210 sec. Inthe “no DTT” test, the same procedure was followed except that no DTTwas added. Table 34 sets forth the results of this experiment using the210 sec. readings. FIGS. 15 and 16 further illustrate the results ofthis test.

TABLE 34 Effect of DTT on PO/HRP pyruvate detection system WITHOUT DTTWITH DTT Blank 0.0021 −0.0021 Factor 2212 −11765   50 μmol/L Standard51.5 35.3   25 μmol/L Standard 24.1 0.0 12.5 μmol/L Standard 9.7 −21.2Delta change at 30 sec. (50 μmol std.) 0.0643 −0.0062 Delta change at 90sec. (50 μmol std.) 0.0890 −0.0103 Delta change at 120 sec. (50 μmolstd.) 0.0903 −0.0113 Delta change at 210 sec. (50 μmol std.) 0.0906−0.0113

These data clearly demonstrate the necessity of the separate reductionstep described in the “second embodiment” of WO 00/28071. The presenceof the reductant interferes with the pyruvate oxidase detection systemused to assay for pyruvate. Therefore, the data presented here confirmthat the time-consuming separate off-line reduction step is essential inthe “second embodiment.” In addition, the off-line reduction stepdescribed in WO 00/28071 will likely elevate significantly the assaycomplexity and cost relative to the present invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. The method of determining the amount of homocysteine in a samplecomprising the steps of: (a) contacting said sample with an enzyme and asubstrate, said enzyme operable to convert homocysteine to cystathioninein the presence of said substrate; (b) contacting said sample with asecond enzyme that reconverts cystathionine to homocysteine with theattendant release of by-products; and (c) determining the amount ofhomocysteine in said sample by measuring the amount of by-productsproduced by the conversion of cystathionine to homocysteine or bymeasuring the amount of substrate consumed, wherein said conversion ofhomocysteine to cystathionine and reconversion of cystathionine tohomocysteine is complete within about 15 minutes.
 2. The method of claim1, said first enzyme being cystathionine β-synthase.
 3. The method ofclaim 1, said substrate being L-serine.
 4. The method of claim 1, saidsecond enzyme being cystathionine β-lyase.
 5. The method of claim 1,said by-products being selected from the group consisting of ammonia,pyruvate, and combinations thereof.
 6. The method of claim 1, includingthe step of repeating steps (a) and (b) a plurality of times.
 7. Themethod of claim 5, said sample further containing lactate dehydrogenaseor a derivative thereof and reduced nicotinamide cofactor or derivativethereof, said pyruvate being measured by the oxidation of said reducednicotinamide cofactor to oxidized nicotinamide cofactor.
 8. The methodof claim 7, said amount of oxidized nicotinamide cofactor being measuredby monitoring said sample at 340 nm.
 9. The method of claim 5, saidsample further comprising pyruvate oxidase, horseradish peroxidase,hydrogen peroxide, and a chromogen, said pyruvate being measured basedon the intensity of an observed color in a colorimetric reaction. 10.The method of claim 5, said ammonia being measured by an ammonia sensor.